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Epitranscriptomics in liver disease: Basic concepts and therapeutic potential

  • Author Footnotes
    † These authors contributed equally.
    Zhicong Zhao
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

    Department of Systems Biology, Beckman Research Institute of City of Hope, Monrovia, CA 91016, USA
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  • Author Footnotes
    † These authors contributed equally.
    Jiaxiang Meng
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
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  • Rui Su
    Affiliations
    Department of Systems Biology, Beckman Research Institute of City of Hope, Monrovia, CA 91016, USA
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  • Jun Zhang
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Shanghai Institute of Digestive Disease, Shanghai 200001, China
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  • Jianjun Chen
    Affiliations
    Department of Systems Biology, Beckman Research Institute of City of Hope, Monrovia, CA 91016, USA
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  • Xiong Ma
    Correspondence
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Shanghai Institute of Digestive Disease, Shanghai 200001, China. Tel.: +86-21-63200874.
    Affiliations
    Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University; Shanghai Institute of Digestive Disease, Shanghai 200001, China
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  • Qiang Xia
    Correspondence
    Corresponding authors. Addresses: Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200127, China. Tel.: +86-21-58752345.
    Affiliations
    Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
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  • Author Footnotes
    † These authors contributed equally.
Open AccessPublished:April 21, 2020DOI:https://doi.org/10.1016/j.jhep.2020.04.009

      Summary

      The development of next-generation sequencing technology and the discovery of specific antibodies targeting chemically modified nucleotides have paved the way for a new era of epitranscriptomics. Cellular RNA is known to dynamically and reversibly undergo different chemical modifications after transcription, such as N6-methyladenosine (m6A), N1-methyladenosine, N6,2′-O-dimethyladenosine, 5-methylcytosine, and 5-hydroxymethylcytidine, whose identity and location comprise the field of epitranscriptomics. Dynamic post-transcriptional modifications determine the fate of target RNAs by regulating various aspects of their processing, including RNA export, transcript processing, splicing, and degradation. The most abundant internal mRNA modification in eukaryotic cells is m6A, which exhibits essential roles in physiological processes, such as embryogenesis, carcinogenesis, and neurogenesis. m6A is deposited by the m6A methyltransferase complex (composed of METTL3/14/16, WTAP, KIAA1429, and RBM15/15B), erased by demethylases (FTO and ALKBH5), and recognised by binding proteins (e.g., YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3). The liver is the largest digestive and metabolic organ, and m6A modifications play unique roles in critical physiological hepatic functions and various liver diseases. This review focuses on the biological roles of m6A RNA methylation in lipid metabolism, viral hepatitis, non-alcoholic fatty liver disease, liver cancer, and tumour metastasis. In addition, we summarise the existing inhibitors targeting m6A regulators and discuss the potential of modulating m6A modifications as a therapeutic strategy.

      Keywords

      Introduction

      Epigenomics describes several regulatory mechanisms that can influence gene expression in response to environmental changes, and a variety of these regulatory processes, such as DNA methylation, histone modification, and chromatin remodelling, have been reported to affect liver physiology and disease.
      • Mann D.A.
      Epigenetics in liver disease.
      Considerable effort has been made to reveal the regulatory mechanisms and functions of RNA modifications, with the term ‘epitranscriptome’ coined in 2012 to describe the chemical RNA modifications that control its structure and function without affecting its sequence.
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      • Meyer K.
      • Korlach J.
      • Vilfan I.D.
      • Jaffrey S.
      • Mason C.E.
      The birth of the epitranscriptome: deciphering the function of RNA modifications.
      ,
      • Helm M.
      • Motorin Y.
      Detecting RNA modifications in the epitranscriptome: predict and validate.
      Thus, the epigenome, epitranscriptome, and epiproteome (PTM) represent our current understanding of gene regulation at the DNA, RNA, and protein level, respectively.
      • Saletore Y.
      • Meyer K.
      • Korlach J.
      • Vilfan I.D.
      • Jaffrey S.
      • Mason C.E.
      The birth of the epitranscriptome: deciphering the function of RNA modifications.
      Recent years have witnessed huge advances in the development of next-generation sequencing (NGS) for distinct epitranscriptomic marks, while scientists have made major breakthroughs in mapping transcriptome-wide RNA modifications.
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      • Li K.
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      • Xiong X.
      • et al.
      Landscape and regulation of mA and mAm methylome across human and mouse tissues.
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      • et al.
      Transcriptome-wide mapping reveals reversible and dynamic N1-methyladenosine methylome.
      • Li X.
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      • Zhou J.
      • et al.
      Base-resolution mapping reveals distinct m1A methylome in nuclear- and mitochondrial-Encoded transcripts.
      In addition, a variety of enzymes have been discovered that can add, remove, or recognise these RNA tags, making these RNA modifications dynamic and reversible. To date, more than 100 different chemical RNA modifications have been identified,
      • Boccaletto P.
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      • Purta E.
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      • Wirecki T.K.
      • et al.
      MODOMICS: a database of RNA modification pathways. 2017 update.
       including N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), 5-methylcytosine (m5C), and 5-hydroxymethylcytidine (hm5C). These RNA modifications have been found in multiple RNA classes, such as mRNA, rRNA, tRNA, and non-coding RNA, with increasing evidence suggesting that they play important roles in post-transcriptional gene regulation. m6A is the most abundant form of internal RNA modification in eukaryotic cells and is related to almost every step of RNA metabolism. It refers to any events in the entire life cycle of RNA molecules, such as processing, transporting, translation, degradation, and so on.
      • Dai D.
      • Wang H.
      • Zhu L.
      • Jin H.
      • Wang X.
      N6-methyladenosine links RNA metabolism to cancer progression.
      Accumulating evidence has suggested that m6A modifications are involved in physiological processes including embryogenesis,
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      • Wang X.
      • Beadell A.V.
      • Lu Z.
      • Shi H.
      • Kuuspalu A.
      • et al.
      m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition.
      carcinogenesis,
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      • Zhang W.
      • Liu Q.
      • Wang L.
      • Ramirez-Moya J.
      • et al.
      mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis.
      and neurogenesis.
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      • Ringeling F.R.
      • Vissers C.
      • Jacob F.
      • Pokrass M.
      • Jimenez-Cyrus D.
      • et al.
      Temporal control of mammalian Cortical neurogenesis by m6A methylation.
      Herein, we briefly describe the regulatory mechanisms and functions of major RNA modifications, such as m6A, m6Am, m1A, m5C, and hm5C, in the epitranscriptome of eukaryotic cells. We also highlight the biological and clinical roles of m6A modification in liver diseases such as viral hepatitis, lipid metabolism disorder, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver cancer. Finally, we summarise the potential application of m6A inhibitors in treating liver diseases. Although a variety of m6A inhibitors have been identified, further studies are required to determine the specificity and side effects of these drugs in treating liver diseases using mouse models. In addition, the combination of m6A inhibitors with existing medical approaches may lead to more effective therapies.

      Epitranscriptomic regulatory mechanisms

      Epitranscriptomic modifications play an essential role in RNA metabolism and can affect gene expression in a post-transcriptional manner. Recent advances in NGS have made it considerably easier to detect and map RNA chemical modification sites and thus have expanded our understanding of epitranscriptomics. Under the precise control of these modifications, target RNAs undergo different fates according to their temporal and spatial context. The biological consequences of m6A modifications have been well-studied; however, the roles and underlying mechanisms of other RNA modifications such as m1A, m6Am, m5C, and hm5C remain largely unknown. Herein, we briefly summarise the important epitranscriptomic mechanisms that regulate RNA metabolism with a focus on m6A RNA methylation (summary in Table 1).
      Table 1Epitranscriptomic regulatory mechanisms in RNA metabolism.
      TypeRegulatorFunction in RNA metabolismYearRefs.
      mRNA exportmRNA translationmRNA decaymRNA stabilitymRNA splicingmiRNA processing
      m6A
       WriterMETTL3++++2013, 2018, 2014, 2015
      • Choe J.
      • Lin S.
      • Zhang W.
      • Liu Q.
      • Wang L.
      • Ramirez-Moya J.
      • et al.
      mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis.
      ,
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
      ,
      • Fustin J.M.
      • Doi M.
      • Yamaguchi Y.
      • Hida H.
      • Nishimura S.
      • Yoshida M.
      • et al.
      RNA-methylation-dependent RNA processing controls the speed of the circadian clock.
      ,
      • Alarcón C.R.
      • Lee H.
      • Goodarzi H.
      • Halberg N.
      • Tavazoie S.F.
      N6-methyladenosine marks primary microRNAs for processing.
      METTL14+2017
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      WTAP+2014
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
       EraserFTO+2017
      • Bartosovic M.
      • Molares H.C.
      • Gregorova P.
      • Hrossova D.
      • Kudla G.
      • Vanacova S.
      N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing.
      ALKBH5-2013
      • Zheng G.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
       ReaderYTHDF1+2015
      • Wang X.
      • Zhao B.S.
      • Roundtree I.A.
      • Lu Z.
      • Han D.
      • Ma H.
      • et al.
      N(6)-methyladenosine modulates messenger RNA translation efficiency.
      YTHDF2+2016
      • Du H.
      • Zhao Y.
      • He J.
      • Zhang Y.
      • Xi H.
      • Liu M.
      • et al.
      YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex.
      YTHDF3++2017
      • Shi H.
      • Wang X.
      • Lu Z.
      • Zhao B.S.
      • Ma H.
      • Hsu P.J.
      • et al.
      YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA.
      YTHDC1++2017, 2016
      • Roundtree I.A.
      • Luo G.Z.
      • Zhang Z.
      • Wang X.
      • Zhou T.
      • Cui Y.
      • et al.
      YTHDC1 mediates nuclear export of N-methyladenosine methylated mRNAs.
      ,
      • Xiao W.
      • Adhikari S.
      • Dahal U.
      • Chen Y.S.
      • Hao Y.J.
      • Sun B.F.
      • et al.
      Nuclear m(6)A reader YTHDC1 regulates mRNA splicing.
      YTHDC2+2017
      • Hsu P.J.
      • Zhu Y.
      • Ma H.
      • Guo Y.
      • Shi X.
      • Liu Y.
      • et al.
      Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis.
      IGF2BPs+2018
      • Huang H.
      • Weng H.
      • Sun W.
      • Qin X.
      • Shi H.
      • Wu H.
      • et al.
      Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.
      m6Am
       WriterPCIF1-+2019, 2019
      • Boulias K.
      • Toczydłowska-Socha D.
      • Hawley B.R.
      • Liberman N.
      • Takashima K.
      • Zaccara S.
      • et al.
      Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.
      ,
      • Sendinc E.
      • Valle-Garcia D.
      • Dhall A.
      • Chen H.
      • Henriques T.
      • Navarrete-Perea J.
      • et al.
      PCIF1 Catalyzes m6Am mRNA methylation to regulate gene expression.
       EraserFTO2017, 2018
      • Wei J.
      • Liu F.
      • Lu Z.
      • Fei Q.
      • Ai Y.
      • He P.C.
      • et al.
      Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.
      ,
      • Mauer J.
      • Luo X.
      • Blanjoie A.
      • Jiao X.
      • Grozhik A.V.
      • Patil D.P.
      • et al.
      Reversible methylation of m6A in the 5' cap controls mRNA stability.
       Reader?
      m1A
       Writer?
       EraserALKBH32016
      • Li X.
      • Xiong X.
      • Wang K.
      • Wang L.
      • Shu X.
      • Ma S.
      • et al.
      Transcriptome-wide mapping reveals reversible and dynamic N1-methyladenosine methylome.
      FTO2018
      • Wei J.
      • Liu F.
      • Lu Z.
      • Fei Q.
      • Ai Y.
      • He P.C.
      • et al.
      Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.
       ReaderYTHDF2+2019
      • Seo K.W.
      • Kleiner R.E.
      YTHDF2 Recognition of N-methyladenosine (mA)-Modified RNA is associated with transcript destabilization.
      m5C
       WriterNSUN2+2017
      • Yang X.
      • Yang Y.
      • Sun B.F.
      • Chen Y.S.
      • Xu J.W.
      • Lai W.Y.
      • et al.
      5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.
       Eraser?
       ReaderYBX1+2019
      • Yang Y.
      • Wang L.
      • Han X.
      • Yang W.L.
      • Zhang M.
      • Ma H.L.
      • et al.
      RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay.
      YTHDF22019
      • Dai X.
      • Gonzalez G.
      • Li L.
      • Li J.
      • You C.
      • Miao W.
      • et al.
      YTHDF2 binds to 5-methylcytosine in RNA and modulates the maturation of Ribosomal RNA.
      ALYREF+2017
      • Yang X.
      • Yang Y.
      • Sun B.F.
      • Chen Y.S.
      • Xu J.W.
      • Lai W.Y.
      • et al.
      5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.
      “+” means enhance; “-” means suppress; “?” means unknown.
      ALKBH3/5, alkB homolog 3/5; ALYREF, Aly/REF export factor; FTO, Fat mass and obesity-associated gene; IGFBPs, insulin-like growth factor 2 mRNA binding proteins; METTL3/14, methyltransferase-like 3/14; NSUN2, NOP2/Sun RNA methyltransferase family member 2; PCIF1, phosphorylated CTD interacting factor 1; WTAP, Wilms' tumour 1 associating protein; YBX1, Y-box binding protein 1; YTHDC1/2, YTH domain containing 1/2; YTHDF1/2/3, YTH domain family protein 1/2/3.

      Purine methylation (m6A/m6Am/m1A)

      First discovered in the 1970s,
      • Ronald D.
      • Karen F.
      • Fritz R.
      Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells.
      m6A is the most frequent internal RNA modification in mammalian messenger RNA, microRNA, and non-coding RNA.
      • Roundtree I.A.
      • Evans M.E.
      • Pan T.
      • He C.
      Dynamic RNA modifications in gene expression regulation.
      This dynamic and reversible modification is introduced by m6A “writers”, removed by m6A “erasers”, and recognised by m6A “readers”. In mammals, METTL3/14, WTAP, KIAA1429, and RBM15/15B comprised the major m6A writer complex, while METTL16 alone functions as an m6A writer,
      • Bokar J.A.
      • Rath-Shambaugh M.E.
      • Ludwiczak R.
      • Narayan P.
      • Rottman F.
      Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex.
      • Liu J.
      • Yue Y.
      • Han D.
      • Wang X.
      • Fu Y.
      • Zhang L.
      • et al.
      A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.
      • Pendleton K.E.
      • Chen B.
      • Liu K.
      • Hunter O.V.
      • Xie Y.
      • Tu B.P.
      • et al.
      The U6 snRNA m6A methyltransferase METTL16 regulates SAM Synthetase Intron Retention.
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
      • Schwartz S.
      • Mumbach M.R.
      • Jovanovic M.
      • Wang T.
      • Maciag K.
      • Bushkin G.G.
      • et al.
      Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites.
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      whereas the demethylases FTO and ALKBH5 can remove m6A modifications from RNA and thus function as erasers.
      • Jia G.
      • Fu Y.
      • Zhao X.
      • Dai Q.
      • Zheng G.
      • Yang Y.
      • et al.
      N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.
      ,
      • Zheng G.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      The m6A readers YTHDF1/2/3, YTHDC1/2, and IGF2BP1/2/3 are a set of m6A binding proteins that recognise m6A methylation and initiate functional signalling.
      • Wang X.
      • Zhao B.S.
      • Roundtree I.A.
      • Lu Z.
      • Han D.
      • Ma H.
      • et al.
      N6-methyladenosine modulates messenger RNA translation efficiency.
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      • Shi H.
      • Wang X.
      • Lu Z.
      • Zhao B.S.
      • Ma H.
      • Hsu P.J.
      • et al.
      YTHDF3 facilitates translation and decay of N-methyladenosine-modified RNA.
      • Roundtree I.A.
      • Luo G.Z.
      • Zhang Z.
      • Wang X.
      • Zhou T.
      • Cui Y.
      • et al.
      YTHDC1 mediates nuclear export of N-methyladenosine methylated mRNAs.
      • Bailey A.S.
      • Batista P.J.
      • Gold R.S.
      • Chen Y.G.
      • de Rooij D.G.
      • Chang H.Y.
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      The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline.
      • Huang H.
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      Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.
      Previous studies have demonstrated that m6A modification has a conserved consensus motif within different species,
      • Harper J.E.
      • Miceli S.M.
      • Roberts R.J.
      • Manley J.L.
      Sequence specificity of the human mRNA N6-adenosine methylase in vitro.
      and some of those m6A regulators are evolutionarily conserved among eukaryotes. For example, METTL3 has homologs in yeast (IME4P),
      • Clancy M.J.
      • Shambaugh M.E.
      • Timpte C.S.
      • Bokar J.A.
      Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene.
      drosophila (dIME4),
      • Hongay C.F.
      • Orr-Weaver T.L.
      Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis.
      and Arabidopsis (MTA).
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      MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor.
      WTAP is also found conserved in different eukaryotes (MUM2 in yeast,
      • Engebrecht J.
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      • Rose K.
      • Kessel T.
      Yeast meiotic mutants proficient for the induction of ectopic recombination.
      Fl(2)d in drosophila,
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      The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain.
      and FIP37 in Arabidopsis
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      ). Considerable efforts have been made to reveal the biological function of m6A modification in mammals, with accumulating evidence suggesting that m6A modification regulates almost every step of RNA metabolism, including mRNA export, translation, stability, splicing, and miRNA processing.
      m6A modification is the most abundant and well-studied RNA modification in the epitranscriptome and is involved in various aspects of mRNA metabolism, including mRNA export, translation, stability, and splicing.
      Studies have revealed that m6A modifications can promote the export of mRNA from the nucleus to the cytoplasm. For instance, METTL3 knockdown in mouse embryonic fibroblasts delays mRNA export,
      • Fustin J.M.
      • Doi M.
      • Yamaguchi Y.
      • Hida H.
      • Nishimura S.
      • Yoshida M.
      • et al.
      RNA-methylation-dependent RNA processing controls the speed of the circadian clock.
      while ALKBH5 depletion enhances mRNA export.
      • Zheng G.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      YTHDC1 has been found to mediate m6A-mRNA export by interacting with the nuclear export adaptor protein SRSF3.
      • Roundtree I.A.
      • Luo G.Z.
      • Zhang Z.
      • Wang X.
      • Zhou T.
      • Cui Y.
      • et al.
      YTHDC1 mediates nuclear export of N-methyladenosine methylated mRNAs.
      m6A modifications have also been found to enhance mRNA translation in several ways. For instance, METTL3 can promote the translation of a series of oncogenic mRNAs by binding with eIF3h,
      • Choe J.
      • Lin S.
      • Zhang W.
      • Liu Q.
      • Wang L.
      • Ramirez-Moya J.
      • et al.
      mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis.
      while YTHDC2 can enhance the translation efficiency of its target mRNA under certain conditions.
      • Hsu P.J.
      • Zhu Y.
      • Ma H.
      • Guo Y.
      • Shi X.
      • Liu Y.
      • et al.
      Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis.
      YT521-B homology domain family (YTHDF) proteins have been shown to play crucial roles in mRNA metabolism. Briefly, YTHDF1 increases translation efficiency by recruiting translation initiation factors such as eIF3,
      • Wang X.
      • Zhao B.S.
      • Roundtree I.A.
      • Lu Z.
      • Han D.
      • Ma H.
      • et al.
      N(6)-methyladenosine modulates messenger RNA translation efficiency.
      whereas YTHDF2 induces the decay of its target mRNAs by recruiting the CCR4-NOT deadenylase complex.
      • Du H.
      • Zhao Y.
      • He J.
      • Zhang Y.
      • Xi H.
      • Liu M.
      • et al.
      YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex.
      In addition, YTHDF3 can promote mRNA translation by cooperating with YTHDF1 and can also affect YTHDF2-mediated mRNA degradation.
      • Shi H.
      • Wang X.
      • Lu Z.
      • Zhao B.S.
      • Ma H.
      • Hsu P.J.
      • et al.
      YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA.
      Insulin-like growth factor 2 mRNA binding proteins (IGF2BPs, including IGF2BP1/2/3) are a family of conserved RNA-binding proteins that control the fate of mRNAs in mammalian cells.
      • Brodt P.
      Role of the microenvironment in liver metastasis: from pre- to Prometastatic Niches.
      It was reported recently that IGF2BPs actually function as a new family of m6A readers and promote the stability of their target mRNAs by recognising the GG(m6A)C sequence.
      • Huang H.
      • Weng H.
      • Sun W.
      • Qin X.
      • Shi H.
      • Wu H.
      • et al.
      Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.
      Pre-mRNA splicing is an essential gene regulatory process. Previous studies have demonstrated that the intronic deposition of m6A is associated with alternative splicing, while m6A modifications near splice-junctions enhance splicing kinetics.
      • Louloupi A.
      • Ntini E.
      • Conrad T.
      • Ørom U.A.V.
      Transient N-6-Methyladenosine transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency.
      FTO has been shown to regulate alternative splicing via m6A by interacting with SRSF2.
      • Bartosovic M.
      • Molares H.C.
      • Gregorova P.
      • Hrossova D.
      • Kudla G.
      • Vanacova S.
      N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3'-end processing.
      PAR-CLIP analysis demonstrated that METTL3 and WTAP are also associated with alternative splicing during RNA processing,
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
      while the m6A reader YTHDC1 has been found to enhance mRNA splicing by selectively recruiting or inhibiting different pre-mRNA splicing factors.
      • Xiao W.
      • Adhikari S.
      • Dahal U.
      • Chen Y.S.
      • Hao Y.J.
      • Sun B.F.
      • et al.
      Nuclear m(6)A reader YTHDC1 regulates mRNA splicing.
      m6A modification is also related to microRNA biogenesis. For example, METTL3 depletion leads to global miRNA downregulation, while METTL3 can methylate target pri-miRNAs and promote miRNA processing by cooperating with DGCR8 and HNRNPA2B1.
      • Alarcón C.R.
      • Lee H.
      • Goodarzi H.
      • Halberg N.
      • Tavazoie S.F.
      N6-methyladenosine marks primary microRNAs for processing.
      ,
      • Alarcón C.R.
      • Goodarzi H.
      • Lee H.
      • Liu X.
      • Tavazoie S.
      • Tavazoie S.F.
      HNRNPA2B1 is a mediator of m(6)A-dependent nuclear RNA processing events.
      Consistently, METTL14 has also been reported to regulate miRNA processing in an m6A-dependent manner.
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      Several studies have suggested that apart from RNA metabolism, m6A modification also plays an important role in RNA structure switches and RNA editing. RNA-binding proteins regulate many cellular processes by identifying RNA-binding motifs (RBMs). m6A can also control the RNA-structure-dependent accessibility of RBMs and affect RNA-protein interactions.
      • Liu N.
      • Dai Q.
      • Zheng G.
      • He C.
      • Parisien M.
      • Pan T.
      N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.
      ,
      • Liu N.
      • Zhou K.I.
      • Parisien M.
      • Dai Q.
      • Diatchenko L.
      • Pan T.
      N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein.
      Adenosine-to-inosine (A-to-I) editing, catalysed by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes, is the most common post-transcriptional nucleotide modification in humans and is associated with many human pathological and physiological processes.
      • Xu X.
      • Wang Y.
      • Liang H.
      The role of A-to-I RNA editing in cancer development.
      A recent study showed a negative correlation between m6A and A-to-I, which is partly due to the unfavourable interaction between m6A transcripts and ADARs. In addition, depleting m6A modification can increase the association of m6A-depleted transcripts with ADARs, resulting in upregulated A-to-I editing.
      • Xiang J.F.
      • Yang Q.
      • Liu C.X.
      • Wu M.
      • Chen L.L.
      • Yang L.
      N6-Methyladenosines modulate A-to-I RNA editing.
      m6Am was first identified in animal cells and viruses in 1975.
      • Wei C.
      • Gershowitz A.
      • Moss B.
      N6, O2'-dimethyladenosine a novel methylated ribonucleoside next to the 5' terminal of animal cell and virus mRNAs.
      If the first nucleotide after the 7-methylguanosine (m7G) cap of mRNA is 2′-O-dimethyladenosine, it can be further methylated at the N6 position to generate m6Am. The proteins that regulate m6Am have recently been elucidated, with accumulating evidence suggesting that PCIF1 is the N6-adenosine methylase that creates the m6Am modification,
      • Boulias K.
      • Toczydłowska-Socha D.
      • Hawley B.R.
      • Liberman N.
      • Takashima K.
      • Zaccara S.
      • et al.
      Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.
      ,
      • Sendinc E.
      • Valle-Garcia D.
      • Dhall A.
      • Chen H.
      • Henriques T.
      • Navarrete-Perea J.
      • et al.
      PCIF1 Catalyzes m6Am mRNA methylation to regulate gene expression.
      while FTO was reported to play an important role in both m6Am and m6A cap demethylation.
      • Wei J.
      • Liu F.
      • Lu Z.
      • Fei Q.
      • Ai Y.
      • He P.C.
      • et al.
      Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.
      ,
      • Mauer J.
      • Luo X.
      • Blanjoie A.
      • Jiao X.
      • Grozhik A.V.
      • Patil D.P.
      • et al.
      Reversible methylation of m6A in the 5' cap controls mRNA stability.
      Like m6A, m6Am is also related to mRNA metabolism. It has been reported that a subset of m6Am initiated transcripts are much more stable than other mRNAs due to their resistance to the mRNA decapping enzyme DCP2
      • Mauer J.
      • Luo X.
      • Blanjoie A.
      • Jiao X.
      • Grozhik A.V.
      • Patil D.P.
      • et al.
      Reversible methylation of m6A in the 5' cap controls mRNA stability.
      ; however, another study suggested that m6Am can reduce cap-dependent translation but has no effect on mRNA stability.
      • Sendinc E.
      • Valle-Garcia D.
      • Dhall A.
      • Chen H.
      • Henriques T.
      • Navarrete-Perea J.
      • et al.
      PCIF1 Catalyzes m6Am mRNA methylation to regulate gene expression.
      Transcriptome-wide m6Am mapping in human tissues indicated a negative correlation between m6Am modified RNA and protein levels.
      • Liu J.
      • Li K.
      • Cai J.
      • Zhang M.
      • Zhang X.
      • Xiong X.
      • et al.
      Landscape and regulation of mA and mAm methylome across human and mouse tissues.
      Therefore, future studies should investigate the role of m6Am modification in various physiological and pathological processes and identify new m6Am readers that directly regulate RNA metabolism.
      N1-methyladenosine (m1A) is typically found in mitochondrial tRNAs and rRNAs, and new m1A-seq techniques have helped demonstrate that m1A also presents at a low abundance in mRNA. Moreover, it has been shown that m1A is enriched within 5ʹ untranslated regions (UTRs) and in the region around start codons.
      • Li X.
      • Xiong X.
      • Wang K.
      • Wang L.
      • Shu X.
      • Ma S.
      • et al.
      Transcriptome-wide mapping reveals reversible and dynamic N1-methyladenosine methylome.
      Since m1A was first identified in tRNA, tRNA m1A methyltransferases have been well-studied, with some known to also introduce m1A into mRNA transcripts.
      • Li X.
      • Xiong X.
      • Zhang M.
      • Wang K.
      • Chen Y.
      • Zhou J.
      • et al.
      Base-resolution mapping reveals distinct m1A methylome in nuclear- and mitochondrial-Encoded transcripts.
      In mRNA, m1A modifications can be removed by both ALKBH3 and FTO,
      • Li X.
      • Xiong X.
      • Wang K.
      • Wang L.
      • Shu X.
      • Ma S.
      • et al.
      Transcriptome-wide mapping reveals reversible and dynamic N1-methyladenosine methylome.
      ,
      • Wei J.
      • Liu F.
      • Lu Z.
      • Fei Q.
      • Ai Y.
      • He P.C.
      • et al.
      Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.
      while YTHDF2 can recognise m1A-modified sites and promote the degradation of target RNAs
      • Seo K.W.
      • Kleiner R.E.
      YTHDF2 Recognition of N-methyladenosine (mA)-Modified RNA is associated with transcript destabilization.
      ; however, future studies should focus on identifying more mRNA-specific m1A methyltransferases and m1A readers.

      Pyrimidine methylation (m5C/hm5C)

      5-methylcytosine (m5C) is a post-transcriptional modification of tRNA, rRNA, and mRNA, with transcriptome-wide mapping showing that m5C sites are mainly enriched in CG-rich regions and distributed in coding sequence regions.
      • Yang X.
      • Yang Y.
      • Sun B.F.
      • Chen Y.S.
      • Xu J.W.
      • Lai W.Y.
      • et al.
      5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.
      It was recently found that m5C-mRNA is mainly introduced by the methyltransferase NSUN2 and is recognised by ALYREF and YBX1.
      • Yang X.
      • Yang Y.
      • Sun B.F.
      • Chen Y.S.
      • Xu J.W.
      • Lai W.Y.
      • et al.
      5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.
      ,
      • Yang Y.
      • Wang L.
      • Han X.
      • Yang W.L.
      • Zhang M.
      • Ma H.L.
      • et al.
      RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay.
      m5C has also been reported to play an important role in regulating RNA metabolism. For instance, ALYREF can regulate mRNA export in a m5C-dependent manner, while NSUN2 knockdown significantly increases nuclear mRNA content in HeLa cells.
      • Yang X.
      • Yang Y.
      • Sun B.F.
      • Chen Y.S.
      • Xu J.W.
      • Lai W.Y.
      • et al.
      5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.
      YBX1 can promote the stability of its target mRNA by recognising m5C-modified sites in cooperation with the mRNA stabiliser PABPC1A,
      • Yang Y.
      • Wang L.
      • Han X.
      • Yang W.L.
      • Zhang M.
      • Ma H.L.
      • et al.
      RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay.
      whereas YTHDF2 affects pre-rRNA processing by binding directly to m5C sites on target RNAs.
      • Dai X.
      • Gonzalez G.
      • Li L.
      • Li J.
      • You C.
      • Miao W.
      • et al.
      YTHDF2 binds to 5-methylcytosine in RNA and modulates the maturation of Ribosomal RNA.
      Although it remains unclear whether FTO or other demethylases can remove m5C sites, recent studies have shown that m5C-modified sites can be oxidised to 5-hydroxymethylcytidine (hm5 C) by Ten-eleven translocation family proteins in mammalian RNA,
      • Fu L.
      • Guerrero C.R.
      • Zhong N.
      • Amato N.J.
      • Liu Y.
      • Liu S.
      • et al.
      Tet-mediated formation of 5-hydroxymethylcytosine in RNA.
      yet our understanding of hm5C RNA modifications remains incomplete.

      m6A RNA methylation in liver: Physiology and pathology

      Of the various modifications to mRNA, m6A is by far the most abundant and well-studied. Expression changes in m6A regulators have been confirmed to cause obvious pathological and physiological aberrations in liver function. For example, hepatocyte-specific knockout of Mettl3 in mice feeding with high-fat diet (HFD) improved insulin sensitivity and decreased fatty acid synthesis.
      • Xie W.
      • Ma L.L.
      • Xu Y.Q.
      • Wang B.H.
      • Li S.M.
      METTL3 inhibits hepatic insulin sensitivity via N6-methyladenosine modification of Fasn mRNA and promoting fatty acid metabolism.
      Liver-specific Ythdf2 knockout mice developed more advanced liver cancer in a chemically induced hepatocellular carcinoma (HCC) model.
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      Igf2bp2-2 transgenic mice are more prone to develop steatohepatitis and may be more prone to hepatocarcinogenesis, resulting from amplified inflammation.
      • Simon Y.
      • Kessler S.M.
      • Bohle R.M.
      • Haybaeck J.
      • Kiemer A.K.
      The insulin-like growth factor 2 (IGF2) mRNA-binding protein p62/IGF2BP2-2 as a promoter of NAFLD and HCC?.
      ,
      • Kessler S.M.
      • Laggai S.
      • Barghash A.
      • Schultheiss C.S.
      • Lederer E.
      • Artl M.
      • et al.
      IMP2/p62 induces genomic instability and an aggressive hepatocellular carcinoma phenotype.
      Igf2bp2-deficient mice are highly resistant to diet-induced fatty liver.
      • Dai N.
      • Zhao L.
      • Wrighting D.
      • Krämer D.
      • Majithia A.
      • Wang Y.
      • et al.
      IGF2BP2/IMP2-Deficient mice resist obesity through enhanced translation of Ucp1 mRNA and other mRNAs encoding mitochondrial proteins.
      However, as IGF2BPs were only recently identified as m6A readers, some previous findings about the functions of IGF2BPs in the liver did not correlate their functions with the m6A-related pathways; thus, further mechanistic studies are needed (see Fig. 1 for a timeline summary). In the next section, we mainly focus on m6A RNA methylation and summarise its physiological and pathological roles in lipid metabolism, viral hepatitis, NAFLD, liver cancer, and tumour metastasis.
      Combining appropriate m6A inhibitors with existing medical approaches, such as immune checkpoint blockade, chemotherapy, or radiotherapy, may lead to more effective therapies.
      Figure thumbnail gr1
      Fig.1Timeline of the key findings on the role of epitranscriptomics in the liver.
      Summary of the key findings of m6A regulators in lipid metabolism, non-alcoholic fatty liver disease, liver cancer, and tumour metastasis. This figure captures the major findings of our study; however, some important advances have not been incorporated due to space restriction. Solid box indicates that this finding is linked to a functional consequence; dashed box indicates that this finding is at an association level. DC, dendritic cell; EMT, epithelial-mesenchymal transition; FTO, Fat mass and obesity-associated gene; HCC, hepatocellular carcinoma; IGFBP1/2, insulin-like growth factor 2 mRNA binding protein 1/2/3; METTL3, methyltransferase-like 3; miRNA, microRNA; NAFLD, non-alcoholic fatty liver disease; WTAP, Wilms' tumour 1 associating protein; YTHDF1/2/3, YTH domain family protein 1/2/3.

      Physiological effects of m6A modification in liver

      m6A RNA methylation is widespread in different tissues, with the “GRACH” motif (R = G or A; A = m6A; H = A, C, or U) having been identified in human, mouse, and pig livers.
      • Liu J.
      • Li K.
      • Cai J.
      • Zhang M.
      • Zhang X.
      • Xiong X.
      • et al.
      Landscape and regulation of mA and mAm methylome across human and mouse tissues.
      ,
      • He S.
      • Wang H.
      • Liu R.
      • He M.
      • Che T.
      • Jin L.
      • et al.
      mRNA N6-methyladenosine methylation of postnatal liver development in pig.
      ,
      • Luo Z.
      • Zhang Z.
      • Tai L.
      • Zhang L.
      • Sun Z.
      • Zhou L.
      Comprehensive analysis of differences of N-methyladenosine RNA methylomes between high-fat-fed and normal mouse livers.
      In contrast, m6Am peaks have been detected in human and mouse livers and are enriched in the canonical “BCA” motif (B = C, G, or U; A = m6Am).
      • Liu J.
      • Li K.
      • Cai J.
      • Zhang M.
      • Zhang X.
      • Xiong X.
      • et al.
      Landscape and regulation of mA and mAm methylome across human and mouse tissues.
      Transcriptome-wide m6A profiling has revealed that m6A-containing genes overlap considerably in human cell lines and mouse livers, with some m6A peaks in the liver identified as tissue specific. More importantly, m6A-containing regions are enriched for single nucleotide polymorphisms (SNPs), with around 50.2% of m6A-related SNPs in the 3′ UTR region. Moreover, m6A regions in liver tissue are particularly enriched for SNPs related to lipid traits.
      • Liu J.
      • Li K.
      • Cai J.
      • Zhang M.
      • Zhang X.
      • Xiong X.
      • et al.
      Landscape and regulation of mA and mAm methylome across human and mouse tissues.
      Interestingly, mutations can lead to a new m6A modification; however, the consequences of these changes and the correlation between hepatic lipid metabolism remain unclear. A recent functional enrichment analysis in pigs, at 3 different liver developmental stages, found that m6A-modified genes are relevant to growth, development, and metabolic processes. Thus, the liver may be exposed to different stimuli during development that result in the differential m6A methylation of the epitranscriptome.
      • He S.
      • Wang H.
      • Liu R.
      • He M.
      • Che T.
      • Jin L.
      • et al.
      mRNA N6-methyladenosine methylation of postnatal liver development in pig.
      The hepatic cytochrome P450 family (CYP450) plays a major role in drug metabolism, yet their activity displays large inter-individual variation.
      • Almazroo O.A.
      • Miah M.K.
      • Venkataramanan R.
      Drug metabolism in the liver.
      It was recently discovered that m6A RNA methylation influences CYP450 expression, with CYP450 2C8 (CYP2C8) expression upregulated by METTL3/14 knockdown, downregulated by FTO knockdown in vitro, and upregulated by YTHDC2 in an m6A-independent manner.
      • Nakano M.
      • Ondo K.
      • Takemoto S.
      • Fukami T.
      • Nakajima M.
      Methylation of adenosine at the N(6) position post-transcriptionally regulates hepatic P450s expression.
      However, more studies are required to determine the effect of m6A modifications on drug metabolism in vivo.
      Circadian rhythms research has recently attracted increasing attention from many scientists. Approximately 10% of liver genes have been identified as rhythmic, i.e., they are mainly driven by the circadian clock; however, only a fifth of these are related to de novo transcription.
      • Koike N.
      • Yoo S.H.
      • Huang H.C.
      • Kumar V.
      • Lee C.
      • Kim T.K.
      • et al.
      Transcriptional architecture and chromatin landscape of the core circadian clock in mammals.
      Some circadian clock-dependent pathways have been reported to mediate hepatic functions, such as glucose metabolism, lipid metabolism, and bile acid synthesis.
      • Mukherji A.
      • Bailey S.M.
      • Staels B.
      • Baumert T.F.
      The circadian clock and liver function in health and disease.
      Moreover, recent studies have demonstrated that m6A mRNA methylation is involved in the regulation of the circadian clock in murine livers, with METTL3 depletion inhibiting RNA processing efficiency and leading to circadian period elongation.
      • Fustin J.M.
      • Doi M.
      • Yamaguchi Y.
      • Hida H.
      • Nishimura S.
      • Yoshida M.
      • et al.
      RNA-methylation-dependent RNA processing controls the speed of the circadian clock.
      In addition, the deletion of Bmal1, an important component of the circadian clock gene regulatory network, not only influences the METTL3 and YTHDF2 proteins but also alters mRNA methylation patterns in murine livers.
      • Zhong X.
      • Yu J.
      • Frazier K.
      • Weng X.
      • Li Y.
      • Cham C.M.
      • et al.
      Circadian clock regulation of hepatic lipid metabolism by modulation of mA mRNA methylation.
      These findings highlight the role that RNA methylation may play in circadian clock regulation under physiological conditions. However, further evidence is needed to support whether the circadian clock can mediate hepatic functions via m6A modification.

      Liver immunology

      The liver is the largest digestive and metabolic organ, and from an immunological perspective it is enriched for various innate immune cell types, such as macrophages, innate lymphoid cells, natural killer (NK) cells, dendritic cells (DCs), and mucosal-associated invariant T cells, which play essential roles in liver homeostasis.
      • Heymann F.
      • Tacke F.
      Immunology in the liver--from homeostasis to disease.
      Liver-resident macrophages (also known as Kupffer cells) are the principal regulator of liver inflammation. Traditionally, macrophages are classified as either M1 (pro-inflammatory), M2 (anti-inflammatory), or Mreg (immunosuppressive), with the extent of hepatic inflammation and repair mainly determined by M1-M2 polarisation.
      • Epelman S.
      • Lavine K.J.
      • Randolph G.J.
      Origin and functions of tissue macrophages.
      In vitro studies have shown that METTL3 drives the M1 polarisation of mouse macrophages by targeting Stat1 mRNA, while METTL3 knockdown markedly enhances M2 polarisation.
      • Liu Y.
      • Liu Z.
      • Tang H.
      • Shen Y.
      • Gong Z.
      • Xie N.
      • et al.
      The N-methyladenosine (m6A)-forming enzyme METTL3 facilitates M1 macrophage polarization through the methylation of mRNA.
      Another study found that YTHDF2 depletion can increase Map2k4 and M4p4k4 mRNA expression to activate NF-κB pathways and promote the inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.
      • Yu R.
      • Li Q.
      • Feng Z.
      • Cai L.
      • Xu Q.
      m6A reader YTHDF2 regulates LPS-induced inflammatory response.
      Besides macrophages, m6A modification has also been associated with DC activation, with METTL3 depletion in DCs resulting in their impaired function. Moreover, METTL3 promotes CD40, CD80, and TIRAP (TLR4 signalling adaptor) translation in DCs and enhances T cell activation in vitro,
      • Wang H.
      • Hu X.
      • Huang M.
      • Liu J.
      • Gu Y.
      • Ma L.
      • et al.
      Mettl3-mediated mRNA m6A methylation promotes dendritic cell activation.
      while YTHDF1-deficient mice show slower growth of B16-OVA tumours and prolonged survival, as the loss of YTHDF1 in DCs enhances the cross-presentation of tumour antigens to CD8+ T cells by inhibiting the translation of lysosomal cathepsins in vivo.
      • Han D.
      • Liu J.
      • Chen C.
      • Dong L.
      • Liu Y.
      • Chang R.
      • et al.
      Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells.
      In addition, METTL3 deletion in murine T cells affects their homeostasis and differentiation by targeting the IL-7/STAT5/SOCS pathway.
      • Li H.B.
      • Tong J.
      • Zhu S.
      • Batista P.J.
      • Duffy E.E.
      • Zhao J.
      • et al.
      m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.
      Consistently, expression of the Socs gene family has also been found to increase in Mettl3−/− Treg cells, decreasing the suppressive function of Treg cells via IL-2/STAT5
      • Tong J.
      • Cao G.
      • Zhang T.
      • Sefik E.
      • Amezcua Vesely M.C.
      • Broughton J.P.
      • et al.
      m6A mRNA methylation sustains Treg suppressive functions.
      (Fig. 2). Since dynamic m6A modifications in macrophages, DCs, and T cells can clearly influence the innate immune response and antitumor immunity, the epitranscriptomic analysis of peripheral or liver-resident immune cells may serve as a biomarker to predict the effect of cancer immunotherapies. Unfortunately, most of these findings have not been considered in a liver-specific environment; thus, the role of immune cell m6A modifications in regulating liver homeostasis and the occurrence of liver diseases remains unclear.
      Figure thumbnail gr2
      Fig. 2The role of m6A modifications in different immune cells.
      In DCs, METTL3 affects CD4+ T cell activation by targeting CD40, CD80, and TIRAP, while YTHDF1 controls the presentation of tumour antigens to CD8+ T cells by influencing the translation of lysosomal cathepsins. In T cells, METTL3 regulates both CD4+ T cell homeostasis and Treg suppressive function by controlling SOCS family gene mRNA levels. In macrophages, YTHDF2 negatively regulates the LPS-induced inflammatory response by targeting NF-κB pathways, while METTL3 drives M1 polarisation by targeting STAT1 mRNA. CTSB/D/L, cathepsin B/D/L; DC, dendritic cell; m6A, N6-methyladenosine; METTL3, methyltransferase-like 3; MTTP, microsomal triglyceride transfer protein; SOCS, suppressor of cytokine signalling; TIRAP, TIR domain containing adaptor protein; YTHDF1/2/3, YTH domain family protein 1/2/3.

      Lipid metabolism, NAFLD, and NASH

      Obesity is regarded as a global human health risk, with NAFLD-NASH-HCC progression being the hepatic consequence of obesity and metabolic syndrome.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      Around 20–30% of patients with liver steatosis may develop NASH, while 30% of patients with NASH may finally progress to cirrhosis or HCC.
      • Younossi Z.M.
      • Loomba R.
      • Rinella M.E.
      • Bugianesi E.
      • Marchesini G.
      • Neuschwander-Tetri B.A.
      • et al.
      Current and future therapeutic regimens for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      Recent studies have investigated the role of m6A RNA methylation in disorders of hepatic lipid metabolism, showing that hyper-methylated m6A sites in HFD-induced fatty livers are enriched for lipid-associated pathway processes, while hypo-methylated m6A sites are associated with translation-associated processes.
      • Luo Z.
      • Zhang Z.
      • Tai L.
      • Zhang L.
      • Sun Z.
      • Zhou L.
      Comprehensive analysis of differences of N-methyladenosine RNA methylomes between high-fat-fed and normal mouse livers.
      m6A-seq in adult porcine livers found highly methylated genes to be enriched in pathways related to ‘positive regulation of the metabolic process’ and ‘fatty acid transport’, suggesting that dynamic m6A methylation may play an essential role in liver metabolism.
      • He S.
      • Wang H.
      • Liu R.
      • He M.
      • Che T.
      • Jin L.
      • et al.
      mRNA N6-methyladenosine methylation of postnatal liver development in pig.
      Consistently, the expression of genes regulating fatty acid synthesis and oxidation (Ehhadh, Fasn, Foxo1, Pgc1aI, and Sirt1) were dramatically decreased in the livers of hepatocyte-specific Mettl3 knockout mice; the improvement in insulin sensitivity and glucose homeostasis induced by METTL3 deletion is also m6A modification dependent.
      • Xie W.
      • Ma L.L.
      • Xu Y.Q.
      • Wang B.H.
      • Li S.M.
      METTL3 inhibits hepatic insulin sensitivity via N6-methyladenosine modification of Fasn mRNA and promoting fatty acid metabolism.
      In addition, the circadian clock also affects hepatic lipid metabolism via YTHDF2-mediated peroxisome proliferator activated receptor-α (PPARα) mRNA decay,
      • Zhong X.
      • Yu J.
      • Frazier K.
      • Weng X.
      • Li Y.
      • Cham C.M.
      • et al.
      Circadian clock regulation of hepatic lipid metabolism by modulation of mA mRNA methylation.
      which provides a new insight into the association between the circadian clock and metabolic diseases.
      m6A modification plays an important role in glucose and lipid homeostasis, while some m6A regulators may be involved in the development of NAFLD and NASH.
      The fat mass and obesity-associated gene (FTO) is known for its effect on adipogenesis and metabolism, with FTO-deficient mice displaying significantly reduced body weight and adipose tissue
      • Fischer J.
      • Koch L.
      • Emmerling C.
      • Vierkotten J.
      • Peters T.
      • Brüning J.C.
      • et al.
      Inactivation of the Fto gene protects from obesity.
      and FTO-overexpressing mice displaying increased food intake.
      • Church C.
      • Moir L.
      • McMurray F.
      • Girard C.
      • Banks G.T.
      • Teboul L.
      • et al.
      Overexpression of Fto leads to increased food intake and results in obesity.
      Studies have also demonstrated that FTO is strongly associated with type 2 diabetes and obesity,
      • Deng X.
      • Su R.
      • Stanford S.
      • Chen J.
      Critical Enzymatic functions of FTO in obesity and cancer.
      and may regulate lipid metabolism in an m6A-dependent manner.
      • Zhao X.
      • Yang Y.
      • Sun B.F.
      • Shi Y.
      • Yang X.
      • Xiao W.
      • et al.
      FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.
      ,
      • Kang H.
      • Zhang Z.
      • Yu L.
      • Li Y.
      • Liang M.
      • Zhou L.
      FTO reduces mitochondria and promotes hepatic fat accumulation through RNA demethylation.
      FTO overexpression in HepG2 cells has been shown to increase the expression of lipid metabolism genes (FASN, SCD1, MGAT1) but decrease the expression of lipid transport genes (MTTP, APOB, LIPC), resulting in lipid accumulation. The FTO R316A mutant, which lacks demethylase activity, did not exert these effects.
      • Kang H.
      • Zhang Z.
      • Yu L.
      • Li Y.
      • Liang M.
      • Zhou L.
      FTO reduces mitochondria and promotes hepatic fat accumulation through RNA demethylation.
      Furthermore, FTO overexpression can upregulate SREBP1c and CIDEC in hepatocytes, both of which are key regulatory factors in lipogenesis,
      • Chen A.
      • Chen X.
      • Cheng S.
      • Shu L.
      • Yan M.
      • Yao L.
      • et al.
      FTO promotes SREBP1c maturation and enhances CIDEC transcription during lipid accumulation in HepG2 cells. Biochimica et biophysica acta.
      while FTO expression is significantly higher in the livers of NAFLD rats and patients with NASH.
      • Guo J.
      • Ren W.
      • Li A.
      • Ding Y.
      • Guo W.
      • Su D.
      • et al.
      Fat mass and obesity-associated gene enhances oxidative stress and lipogenesis in nonalcoholic fatty liver disease.
      ,
      • Lim A.
      • Zhou J.
      • Sinha R.A.
      • Singh B.K.
      • Ghosh S.
      • Lim K.H.
      • et al.
      Hepatic FTO expression is increased in NASH and its silencing attenuates palmitic acid-induced lipotoxicity.
      More importantly, FTO knockdown using shRNA significantly alleviated dexamethasone-induced fatty liver in mice.
      • Hu Y.
      • Feng Y.
      • Zhang L.
      • Jia Y.
      • Cai D.
      • Qian S.B.
      • et al.
      GR-mediated FTO transactivation induces lipid accumulation in hepatocytes via demethylation of mA on lipogenic mRNAs.
      Taken together, these findings suggest that FTO may play a deleterious role in hepatocytes under lipotoxic conditions by altering m6A modifications (Fig. 3).
      Figure thumbnail gr3
      Fig. 3The role of m6A modifications in hepatic lipid metabolism.
      METTL3, FTO, and YTHDF2 regulate hepatic lipid metabolism by altering the RNA methylation of genes involved in fatty acid oxidation, lipogenesis, and lipid transport. METTL3 also regulates hepatic insulin sensitivity via its m6A methylase activity. The circadian clock affects lipid metabolism via the METTL3/m6A/YTHDF2/PPARα decay pathway. APOB, apolipoprotein B; CIDEC, cell death inducing DFFA like effector c; Ehhadh, enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase; FASN, fatty acid synthase; Foxo1, forkhead box O1; FTO, Fat mass and obesity-associated gene; LPC, lipase C; METTL3, methyltransferase-like 3; MGAT1, alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase; MTTP, microsomal triglyceride transfer protein; PGC1α, PPARG coactivator 1 α; PPARα, peroxisome proliferator activated receptor-α; SCD1, stearoyl-CoA desaturase-1; SIRT1, sirtuin 1; SREBP1, sterol regulatory element-binding protein 1; YTHDF1/2/3, YTH domain family protein 1/2/3.
      Insulin-like growth factor 2 (IGF2) plays a pivotal role in mammalian growth by regulating metabolism
      • Nielsen F.C.
      The molecular and cellular biology of insulin-like growth factor II.
      ; however, the role of IGF2BPs in hepatic lipid metabolism remains controversial. Liver-specific IGF2BP2-2 (splice variant IMP2-2/p62) overexpression has been shown to induce steatosis in mice by attenuating PTEN expression and increasing AKT activation downstream.
      • Tybl E.
      • Shi F.D.
      • Kessler S.M.
      • Tierling S.
      • Walter J.
      • Bohle R.M.
      • et al.
      Overexpression of the IGF2-mRNA binding protein p62 in transgenic mice induces a steatotic phenotype.
      Consistently, global Igf2bp2/Imp2 knockout mice are resistant to diet-induced fatty liver disease due to upregulated Ucp1 mRNA translation,
      • Dai N.
      • Zhao L.
      • Wrighting D.
      • Krämer D.
      • Majithia A.
      • Wang Y.
      • et al.
      IGF2BP2/IMP2-Deficient mice resist obesity through enhanced translation of Ucp1 mRNA and other mRNAs encoding mitochondrial proteins.
      while Igf2bp2-2 has been shown to promote NASH development and may also drive progression from NAFLD to HCC in mouse models.
      • Simon Y.
      • Kessler S.M.
      • Bohle R.M.
      • Haybaeck J.
      • Kiemer A.K.
      The insulin-like growth factor 2 (IGF2) mRNA-binding protein p62/IGF2BP2-2 as a promoter of NAFLD and HCC?.
      ,
      • Simon Y.
      • Kessler S.M.
      • Gemperlein K.
      • Bohle R.M.
      • Müller R.
      • Haybaeck J.
      • et al.
      Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis.
      Conversely, another study found that HFD-fed hepatocyte-specific Ifg2bp2 knockout mice display increased triglyceride accumulation and modest fatty liver, potentially due to increased PPARα and Cpt1a mRNA degradation.
      • Regué L.
      • Minichiello L.
      • Avruch J.
      • Dai N.
      Liver-specific deletion of IGF2 mRNA binding protein-2/IMP2 reduces hepatic fatty acid oxidation and increases hepatic triglyceride accumulation.
      Although the HFD-fed mouse model has been used to study the role of m6A in NASH and NAFLD, there is still a lack of transcriptome-wide m6A mapping data using human samples. In addition, further studies are needed to elucidate the multi-functional role of m6A regulators during the progression of NAFLD-NASH-HCC axis.

      Hepatitis virus infection

      HBV infection is not only the leading cause of chronic hepatitis, but is also strongly associated with cirrhosis and HCC.
      • Levrero M.
      • Pollicino T.
      • Petersen J.
      • Belloni L.
      • Raimondo G.
      • Dandri M.
      Control of cccDNA function in hepatitis B virus infection.
      Recent studies have suggested that m6A modifications can regulate the HBV life cycle by either reducing the stability of HBV RNA and the expression of HBV proteins, or promoting reverse transcription of pregenomic RNA.
      • Imam H.
      • Khan M.
      • Gokhale N.S.
      • McIntyre A.B.R.
      • Kim G.W.
      • Jang J.Y.
      • et al.
      N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle.
      m6A modifications have also been reported to regulate the HCV life cycle, with METTL3 and METTL14 depletion enhancing HCV infection in Huh7 cells and FTO knockout having the opposite effect. Moreover, YTHDF proteins co-localize with lipid droplets and may regulate HCV particle production via m6A modification during HCV infection.
      • Gokhale N.S.
      • McIntyre A.B.R.
      • McFadden M.J.
      • Roder A.E.
      • Kennedy E.M.
      • Gandara J.A.
      • et al.
      N6-Methyladenosine in Flaviviridae viral RNA genomes regulates infection.
      Interferon-stimulated genes (ISGs) play a vital role in the IFN-dependent antiviral immune response.
      • Ivashkiv L.B.
      • Donlin L.T.
      Regulation of type I interferon responses.
      DDX46, an essential helicase during the early stages of the antiviral immune response, recruits ALKBH5 to demethylate m6A-modified antiviral transcripts and inhibits interferon production.
      • Zheng Q.
      • Hou J.
      • Zhou Y.
      • Li Z.
      • Cao X.
      The RNA helicase DDX46 inhibits innate immunity by entrapping m(6)A-demethylated antiviral transcripts in the nucleus.
      Moreover, viral infection increases the expression of ISGs in vitro following METTL3 depletion, whereas YTHDF3 cooperates with PABP1 and eIF4G2 to suppress ISGs by promoting FOXO3 mRNA under normal conditions.
      • Zhang Y.
      • Wang X.
      • Zhang X.
      • Wang J.
      • Ma Y.
      • Zhang L.
      • et al.
      RNA-binding protein YTHDF3 suppresses interferon-dependent antiviral responses by promoting FOXO3 translation.
      Consistently, Ythdf3-deficient mice display enhanced Ifnb mRNA induction and are more resistant to viral infection.
      • Zhang Y.
      • Wang X.
      • Zhang X.
      • Wang J.
      • Ma Y.
      • Zhang L.
      • et al.
      RNA-binding protein YTHDF3 suppresses interferon-dependent antiviral responses by promoting FOXO3 translation.
      ,
      • Winkler R.
      • Gillis E.
      • Lasman L.
      • Safra M.
      • Geula S.
      • Soyris C.
      • et al.
      m(6)A modification controls the innate immune response to infection by targeting type I interferons.
      Taken together, these findings reveal that m6A modifications play a novel role in antiviral innate immune responses.

      Liver cancer

      Recent studies have revealed that m6A modifications play an important role in human cancer progression.
      • Deng X.
      • Su R.
      • Feng X.
      • Wei M.
      • Chen J.
      Role of N6-methyladenosine modification in cancer.
      • Deng X.
      • Su R.
      • Weng H.
      • Huang H.
      • Li Z.
      • Chen J.
      RNA N6-methyladenosine modification in cancers: current status and perspectives.
      • Huang H.
      • Weng H.
      • Deng X.
      • Chen J.
      RNA modifications in cancer: functions, mechanisms, and therapeutic Implications.
      On the one hand, m6A enzymes can regulate different tumour-promoting genes or tumour suppressor genes. On the other hand, the expression and activity of these m6A enzymes can also be affected by different factors, such as an hypoxic microenvironment and post-translational modifications (PTMs), thereby influencing the function of m6A modification in cancer. For instance, the methyltransferases METTL3 and METTL14 were shown to promote tumour progression in many types of cancers including leukaemia, gastric cancer, breast cancer, colon cancer, bladder cancer, etc.
      • Wang Q.
      • Chen C.
      • Ding Q.
      • Zhao Y.
      • Wang Z.
      • Chen J.
      • et al.
      METTL3-mediated m(6)A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance.
      • Cai X.
      • Wang X.
      • Cao C.
      • Gao Y.
      • Zhang S.
      • Yang Z.
      • et al.
      HBXIP-elevated methyltransferase METTL3 promotes the progression of breast cancer via inhibiting tumor suppressor let-7g.
      • Zhang Y.
      • Kang M.
      • Zhang B.
      • Meng F.
      • Song J.
      • Kaneko H.
      • et al.
      m6A modification-mediated CBX8 induction regulates stemness and chemosensitivity of colon cancer via upregulation of LGR5.
      • Cheng M.
      • Sheng L.
      • Gao Q.
      • Xiong Q.
      • Zhang H.
      • Wu M.
      • et al.
      The m6A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-κB/MYC signaling network.
      • Weng H.
      • Huang H.
      • Wu H.
      • Qin X.
      • Zhao B.S.
      • Dong L.
      • et al.
      METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes Leukemogenesis via mRNA mA modification.
      • Vu L.P.
      • Pickering B.F.
      • Cheng Y.
      • Zaccara S.
      • Nguyen D.
      • Minuesa G.
      • et al.
      The N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells.
      However, in endometrial cancer, attenuated m6A methylation caused by decreased METTL3 expression and a METTL14 loss-of-function mutation leads to enhanced cell proliferation, migration, and invasion.
      • Liu J.
      • Eckert M.A.
      • Harada B.T.
      • Liu S.M.
      • Lu Z.
      • Yu K.
      • et al.
      m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.
      SUMOylation is a reversible PTM process whereby small ubiquitin-like modifier (SUMO) are attached to protein substrates, thus changing the stability and activity of targeted proteins.
      • Geiss-Friedlander R.
      • Melchior F.
      Concepts in sumoylation: a decade on.
      In a human non-small cell lung carcinoma cell line, SUMOylation is reported to influence the activity of METTL3 and enhance tumorigenesis in vitro and in vivo.
      • Du Y.
      • Hou G.
      • Zhang H.
      • Dou J.
      • He J.
      • Guo Y.
      • et al.
      SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function.
      In the next section, we will focus on liver cancer and discuss the function of m6A RNA modification in HCC, intrahepatic cholangiocarcinoma (ICC), and hepatoblastoma (HB).
      Evidence is emerging that m6A modification participates in liver cancer pathogenesis and metastasis by regulating the expression of tumour-related genes.

      Hepatocellular carcinoma

      HCC is one of the most common malignancies worldwide, with the sixth highest incidence among malignant tumours and fourth highest mortality rate according to recent statistics.
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • Siegel R.L.
      • Torre L.A.
      • Jemal A.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      Bioinformatic analyses have revealed that some m6A regulators can be useful for prognostic stratification; for instance, IGF2BP3, YTHDF1, and YTHDF2 increase the risk of the clinical outcome, whereas METTL14 and ZC3H13 exert protective roles.
      • Li Y.
      • Xiao J.
      • Bai J.
      • Tian Y.
      • Qu Y.
      • Chen X.
      • et al.
      Molecular characterization and clinical relevance of m(6)A regulators across 33 cancer types.
      The combination of YTHDF1 and METTL3 may act as a biomarker for evaluating prognosis and reflecting the degree of HCC malignancy.
      • Zhou Y.
      • Yin Z.
      • Hou B.
      • Yu M.
      • Chen R.
      • Jin H.
      • et al.
      Expression profiles and prognostic significance of RNA N6-methyladenosine-related genes in patients with hepatocellular carcinoma: evidence from independent datasets.
      Indeed, it has been reported that total RNA m6A levels are lower in HCC tissue than in adjacent tissue, whereas mRNA m6A levels are significantly higher.
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      ,
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      Considerable efforts have been made to determine the possible roles of m6A modifications in HCC, with accumulating evidence suggesting that m6A writers, erasers, and readers can regulate liver cancer development and progression by targeting various tumour-related genes (Fig. 4). For example, high METTL3 expression is associated with poor HCC prognosis, with its overexpression promoting cell proliferation, migration, and colony formation in an m6A-YTHDF2-dependent manner by regulating SOCS2 mRNA.
      • Chen M.
      • Wei L.
      • Law C.T.
      • Tsang F.H.
      • Shen J.
      • Cheng C.L.
      • et al.
      RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.
      In addition, METTL3 overexpression markedly reduces RDM1 mRNA expression via m6A modifications, which can bind to and enhance the stability of the tumour suppressor p53.
      • Chen S.L.
      • Liu L.L.
      • Wang C.H.
      • Lu S.X.
      • Yang X.
      • He Y.F.
      • et al.
      Loss of RDM1 enhances hepatocellular carcinoma progression via p53 and Ras/Raf/ERK pathways.
      WTAP is highly expressed in HCC tissue and is an independent predictor of survival.
      • Sera T.
      • Hiasa Y.
      • Mashiba T.
      • Tokumoto Y.
      • Hirooka M.
      • Konishi I.
      • et al.
      Wilms' tumour 1 gene expression is increased in hepatocellular carcinoma and associated with poor prognosis.
      ,
      • Chen Y.
      • Peng C.
      • Chen J.
      • Chen D.
      • Yang B.
      • He B.
      • et al.
      WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.
      Mechanistically, it is thought that ETS1 acts as the downstream target of WTAP and enhances the transcription of the cell cycle regulators p21 and p27 in conjunction with the RNA stabiliser HuR.
      • Chen Y.
      • Peng C.
      • Chen J.
      • Chen D.
      • Yang B.
      • He B.
      • et al.
      WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.
      Another report found that lower METTL14 protein expression levels are associated with shorter overall and recurrence-free survival, with METTL14 modulating primary microRNA126 in an m6A-dependent manner.
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      The role of FTO in HCC has recently been reported. FTO is highly expressed in HCC and high expression of FTO is usually related to poor survival in patients with HCC. Moreover, studies have demonstrated that FTO can induce tumorigenesis of HCC by regulating PKM2 in a m6A modification-dependent manner.
      • Li J.
      • Zhu L.
      • Shi Y.
      • Liu J.
      • Lin L.
      • Chen X.
      m6A demethylase FTO promotes hepatocellular carcinoma tumorigenesis via mediating PKM2 demethylation.
      m6A modifications are also involved in cancer cell epithelial-mesenchymal transition (EMT). It has been shown that knockdown of METTL3 can downregulate the expression of Snail, a key EMT transcription factor, and then decrease in vitro invasion as well as EMT in HCC cell lines.
      • Lin X.
      • Chai G.
      • Wu Y.
      • Li J.
      • Chen F.
      • Liu J.
      • et al.
      RNA m(6)A methylation regulates the epithelial mesenchymal transition of cancer cells and translation of Snail.
      Figure thumbnail gr4
      Fig. 4m6A modifications regulate tumour-related genes in HCC.
      m6A regulators (METTL3, METTL14, WTAP, YTHDF2, FTO) control the RNA metabolism of tumour promoter genes, tumour suppressor genes, and ncRNAs that are essential for liver cancer pathogenesis. As IGF2BPs were identified as new m6A readers just recently, those previous findings about the functions of IGF2BP1 in HCC did not correlate their functions with the m6A-related pathways, so a dotted box was used for IGF2BP1-related signalling pathways here. EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; FOXK1, forkhead box K1; FTO, Fat mass and obesity-associated gene; HCC, hepatocellular carcinoma; IGFBP1/2, insulin-like growth factor 2 mRNA binding protein 1/2/3; IL11, interleukin 11; METTL3/14, methyltransferase-like 3/14; ncRNA, non-coding RNA; RDM1, RAD52 motif containing 1; PDLIM7, PDZ And LIM domain 7; PKM2, pyruvate kinase M2; SOCS2, suppressor of cytokine signalling 2; WTAP, Wilms' tumour 1 associating protein; YTHDF1/2/3, YTH domain family protein 1/2/3.
      As m6A readers, YTHDF proteins are involved in HCC tumorigenesis as well. Overexpression of the YTHDF1 is associated with the poor prognosis of patients with HCC,
      • Zhao X.
      • Chen Y.
      • Mao Q.
      • Jiang X.
      • Jiang W.
      • Chen J.
      • et al.
      Overexpression of YTHDF1 is associated with poor prognosis in patients with hepatocellular carcinoma.
      while low YTHDF2 levels can be regarded as an adverse prognosis factor for patients with HCC.
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      ,
      • Zhong L.
      • Liao D.
      • Zhang M.
      • Zeng C.
      • Li X.
      • Zhang R.
      • et al.
      YTHDF2 suppresses cell proliferation and growth via destabilizing the EGFR mRNA in hepatocellular carcinoma.
      Furthermore, recent studies have suggested that microRNA145 downregulates YTHDF2 by targeting its 3′ UTR
      • Yang Z.
      • Li J.
      • Feng G.
      • Gao S.
      • Wang Y.
      • Zhang S.
      • et al.
      MicroRNA-145 modulates N(6)-methyladenosine levels by targeting the 3'-untranslated mRNA region of the N(6)-methyladenosine binding YTH domain family 2 protein.
      and hypoxia specifically suppresses YTHDF2 protein levels in HCC cell lines.
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      ,
      • Zhong L.
      • Liao D.
      • Zhang M.
      • Zeng C.
      • Li X.
      • Zhang R.
      • et al.
      YTHDF2 suppresses cell proliferation and growth via destabilizing the EGFR mRNA in hepatocellular carcinoma.
      YTHDF2 also processes the decay of IL11 and SERPINE2 mRNA, which are important for vessel normalisation and inflammation,
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      and can bind to the 3′ UTR m6A modification site of EGFR and downregulate EGFR mRNA in HCC cells.
      • Zhong L.
      • Liao D.
      • Zhang M.
      • Zeng C.
      • Li X.
      • Zhang R.
      • et al.
      YTHDF2 suppresses cell proliferation and growth via destabilizing the EGFR mRNA in hepatocellular carcinoma.
      IGF2BPs can selectively bind to their target RNAs and regulate tumorigenesis. High IGF2BP1 expression is associated with poor outcomes in HCC
      • Gutschner T.
      • Hämmerle M.
      • Pazaitis N.
      • Bley N.
      • Fiskin E.
      • Uckelmann H.
      • et al.
      Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an important protumorigenic factor in hepatocellular carcinoma.
      and its oncogenic role has been linked mechanistically to its mRNA processing abilities, with IGF2BP1 reported to stabilise c-MYC and MKI67 mRNAs and increase their protein levels in HCC cell lines.
      • Gutschner T.
      • Hämmerle M.
      • Pazaitis N.
      • Bley N.
      • Fiskin E.
      • Uckelmann H.
      • et al.
      Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an important protumorigenic factor in hepatocellular carcinoma.
      Consistently, IGF2BP1 has been shown to promote SRF expression in an m6A-dependent manner and enhance tumour cell growth and invasion in vitro.
      • Muller S.
      • Glass M.
      • Singh A.K.
      • Haase J.
      • Bley N.
      • Fuchs T.
      • et al.
      IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner.
      IGF2BP1 can also interact with LINC01093 and facilitate GLI1 mRNA decay in HCC cells,
      • He J.
      • Zuo Q.
      • Hu B.
      • Jin H.
      • Wang C.
      • Cheng Z.
      • et al.
      A novel, liver-specific long noncoding RNA LINC01093 suppresses HCC progression by interaction with IGF2BP1 to facilitate decay of GLI1 mRNA.
      while LINC01138 is stabilised by IGF2BP1/IGF2BP3 and associated with poor HCC outcomes.
      • Li Z.
      • Zhang J.
      • Liu X.
      • Li S.
      • Wang Q.
      • Chen Di
      • et al.
      The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma.
      Another study found that IGF2BP1 can destabilise the lncRNA HULC, which is highly upregulated and correlated with liver cancer staging and grading, via the CCR4-NOT deadenylase complex.
      • Hämmerle M.
      • Gutschner T.
      • Uckelmann H.
      • Ozgur S.
      • Fiskin E.
      • Gross M.
      • et al.
      Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1).
      Moreover, the HBV x protein (HBx) has been shown to inhibit microRNA-216b expression and stimulate IGF2BP2 expression in HCC cell lines.
      • Liu F.Y.
      • Zhou S.J.
      • Deng Y.L.
      • Zhang Z.Y.
      • Zhang E.L.
      • Wu Z.B.
      • et al.
      MiR-216b is involved in pathogenesis and progression of hepatocellular carcinoma through HBx-miR-216b-IGF2BP2 signaling pathway.
      IGF2BP3 is highly expressed in human liver tumours
      • Nguyen L.H.
      • Robinton D.A.
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      • Wu L.
      • Li L.
      • Rakheja D.
      • et al.
      Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models.
      and has been reported to act as a sensitive HCC biomarker.
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      • Breuhahn K.
      • Fritzsche F.
      • et al.
      Insulin-like growth factor II mRNA-binding protein 3 (IMP3) expression in hepatocellular carcinoma. A clinicopathological analysis with emphasis on diagnostic value.
      ,
      • Nischalke H.D.
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      • Feldmann G.
      • et al.
      Detection of IGF2BP3, HOXB7, and NEK2 mRNA expression in brush cytology specimens as a new diagnostic tool in patients with biliary strictures.
      Indeed, Igf2bp3 is the downstream oncogenic effector of Lin28b, whose overexpression has been found to drive liver tumorigenesis in murine models.
      • Nguyen L.H.
      • Robinton D.A.
      • Seligson M.T.
      • Wu L.
      • Li L.
      • Rakheja D.
      • et al.
      Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models.
      Several studies have yielded contrasting results. For instance, METTL14 knockdown has been shown to significantly enhance HCC metastasis and METTL14 overexpression, thereby suppressing HCC invasion.
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      Conversely, METTL14 overexpression has also been reported to increase HCC migration and cell proliferation.
      • Chen M.
      • Wei L.
      • Law C.T.
      • Tsang F.H.
      • Shen J.
      • Cheng C.L.
      • et al.
      RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.
      Moreover, it has been shown that hypoxia-induced YTHDF2 downregulation is HIF1α-dependent in HCC cells,
      • Zhong L.
      • Liao D.
      • Zhang M.
      • Zeng C.
      • Li X.
      • Zhang R.
      • et al.
      YTHDF2 suppresses cell proliferation and growth via destabilizing the EGFR mRNA in hepatocellular carcinoma.
      while another study demonstrated that this effect was dependent on HIF2α rather than HIF1α.
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      These contradictory findings illustrate the complicated and multi-functional nature of the roles of m6A regulatory enzymes in HCC.

      Intrahepatic cholangiocarcinoma

      ICC originates from bile duct or hepatic duct epithelial cells and is the second most common primary liver cancer, with a considerably higher mortality rate than HCC.
      • Bridgewater J.
      • Galle P.R.
      • Khan S.A.
      • Llovet J.M.
      • Park J.W.
      • Patel T.
      • et al.
      Guidelines for the diagnosis and management of intrahepatic cholangiocarcinoma.
      ICC has some common clinical features with HCC; however, relatively little is known about the role of m6A modifications in ICC. WTAP overexpression in ICC tissue has been reported to regulate the motility of cholangiocarcinoma cells,
      • Jo H.J.
      • Shim H.E.
      • Han M.E.
      • Kim H.J.
      • Kim K.S.
      • Baek S.
      • et al.
      WTAP regulates migration and invasion of cholangiocarcinoma cells.
      while low FTO expression predicts poor ICC prognosis. Moreover, the mRNA stability of TEAD2, a common oncogene in multiple tumours, is increased by inhibiting FTO in ICC cell lines.
      • Rong Z.X.
      • Li Z.
      • He J.J.
      • Liu L.Y.
      • Ren X.X.
      • Gao J.
      • et al.
      Downregulation of fat mass and obesity associated (FTO) promotes the progression of intrahepatic cholangiocarcinoma.
      However, more studies are required to further elucidate the regulatory network of m6A modifications in ICC.

      Hepatoblastoma

      HB originates from undifferentiated hepatic progenitor cells and is the most common liver cancer of infancy and childhood.
      • Sharma D.
      • Subbarao G.
      • Saxena R.
      Hepatoblastoma.
      A recent transcriptome-wide m6A mapping study in HB found that enhanced m6A mRNA methylation may promote HB proliferation by regulating the Wnt/β-catenin pathway. Consistently, METTL3 may be a potential prognostic biomarker for patients with HB,
      • Liu L.
      • Wang J.
      • Sun G.
      • Wu Q.
      • Ma J.
      • Zhang X.
      • et al.
      m6A mRNA methylation regulates CTNNB1 to promote the proliferation of hepatoblastoma.
      while WTAP, FTO, and YTHDF2 knockdown in HepG2 cells dramatically decreases HB cell proliferation.
      • Liu L.
      • Wang J.
      • Sun G.
      • Wu Q.
      • Ma J.
      • Zhang X.
      • et al.
      m6A mRNA methylation regulates CTNNB1 to promote the proliferation of hepatoblastoma.
      Thus, the mechanisms underlying these m6A regulators should be addressed by future studies.

      Tumour metastasis

      Tumour metastasis is a major barrier to the successful management of gastrointestinal tumours.
      • Brodt P.
      Role of the microenvironment in liver metastasis: from pre- to Prometastatic Niches.
      It is well known that both acellular (e.g. extracellular matrix proteins) and cellular components (e.g. circulating tumour cells, immune cells etc.) are involved in the development or regression of tumour metastases
      • Williamson T.
      • Sultanpuram N.
      • Sendi H.
      The role of liver microenvironment in hepatic metastasis.
      ; however, m6A RNA modifications have also been reported to regulate liver cancer cell metastasis. For example, METTL14 suppresses HCC metastasis by interacting with DGCR8. In both intrahepatic and lung metastasis models, METTL14 depletion in HCC cells increases the number of tumour metastases, suggesting that METTL14 depletion enhances the metastasis of HCC cells.
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      In addition, liver-specific Ythdf2 knockout mice, with chemically induced HCC, have been found to develop more lung metastases,
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      while microRNA126 has been shown to suppress HCC metastatic colonization and angiogenesis by potentially targeting IGF2BP2, PITPNC1, and MERTK.
      • Png K.J.
      • Halberg N.
      • Yoshida M.
      • Tavazoie S.F.
      A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells.
      EMT is the primary method of metastasis, during which tumour cells lose their epithelial markers and gain mesenchymal traits, promoting the migration of tumour cells into the bloodstream and facilitating metastatic dissemination and colonisation.
      • Quail D.F.
      • Joyce J.A.
      Microenvironmental regulation of tumor progression and metastasis.
      METTL3 deficiency suppresses liver cancer cell metastasis in vivo by regulating the translational efficiency of the EMT transcription factor Snail.
      • Lin X.
      • Chai G.
      • Wu Y.
      • Li J.
      • Chen F.
      • Liu J.
      • et al.
      RNA m(6)A methylation regulates the epithelial mesenchymal transition of cancer cells and translation of Snail.
      Hepatic metastases can occur from different primary cancers in the body and are closely correlated with poor survival; indeed, the liver is the dominant metastatic site for primary gastrointestinal tumours.
      • Hess K.R.
      • Varadhachary G.R.
      • Taylor S.H.
      • Wei W.
      • Raber M.N.
      • Lenzi R.
      • et al.
      Metastatic patterns in adenocarcinoma.
      It has been shown that METTL3 overexpression promotes gastric cancer liver metastasis in vitro and in vivo, while METTL3 stimulates the m6A modification of HDGF mRNA and IGF2BP3 enhances mRNA stability. Nuclear HDGF has been found to upregulate GLUT4 and ENO2 expression, which are correlated with tumour liver metastasis.
      • Wang Q.
      • Chen C.
      • Ding Q.
      • Zhao Y.
      • Wang Z.
      • Chen J.
      • et al.
      METTL3-mediated m(6)A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance.
      The METTL3/miR1246/SPRED2 axis also plays an important role in colorectal cancer (CRC) liver metastasis, with METTL3-knockdown CRC cells displaying fewer metastatic nodules in a mouse model of tumour metastasis.
      • Peng W.
      • Li J.
      • Chen R.
      • Gu Q.
      • Yang P.
      • Qian W.
      • et al.
      Upregulated METTL3 promotes metastasis of colorectal cancer via miR-1246/SPRED2/MAPK signaling pathway.
      It has been shown that circNSUN2 levels are upregulated in the serum samples as well as tumour tissues of patients with CRC and liver metastases and predict poor patient survival. Mechanistically, m6A modifications promote the cytoplasmic export of circNSUN2, which stabilises HMGA2 mRNA via the circNSUN2/IGF2BP2/HMGA2 complex, while HMGA2 induces EMT and contributes to CRC liver metastases
      • Chen R.X.
      • Chen X.
      • Xia L.P.
      • Zhang J.X.
      • Pan Z.Z.
      • Ma X.D.
      • et al.
      N(6)-methyladenosine modification of circNSUN2 facilitates cytoplasmic export and stabilizes HMGA2 to promote colorectal liver metastasis.
      (summary in Table 2).
      Table 2Multiple functions of m6A regulator in liver cancer.
      DiseaseTypem6A regulatorExpressionRelated targetFunctionYearRefs.
      HCCWriterMETTL3UpSOCS2METTL3 represses SOCS2 expression via an m6A-YTHDF2-dependent mechanism2018
      • Chen M.
      • Wei L.
      • Law C.T.
      • Tsang F.H.
      • Shen J.
      • Cheng C.L.
      • et al.
      RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.
      HCCWriterMETTL3RDM1METTL3 supresses RDM1 mRNA via m6A modification. RDM1 binds to p53 and enhance its stability2019
      • Chen S.L.
      • Liu L.L.
      • Wang C.H.
      • Lu S.X.
      • Yang X.
      • He Y.F.
      • et al.
      Loss of RDM1 enhances hepatocellular carcinoma progression via p53 and Ras/Raf/ERK pathways.
      HCCWriterWTAPUpETS1WTAP related m6A modification promotes HCC progression through HuR-ETS1-p21/p27 axis2019
      • Chen Y.
      • Peng C.
      • Chen J.
      • Chen D.
      • Yang B.
      • He B.
      • et al.
      WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.
      HCC & TMWriterMETTL14DownmiRNA126METTL14 interacts with DGCR8 and positively modulates the primary miRNA126 process2017
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      HCC & TMWriter

      Reader
      METTL3

      YTHDF1
      Up

      Up
      SnailMETTL3 and YTHDF1 are involved in the epithelial-mesenchymal transition of Snail in HCC cell lines2019
      • Lin X.
      • Chai G.
      • Wu Y.
      • Li J.
      • Chen F.
      • Liu J.
      • et al.
      RNA m(6)A methylation regulates the epithelial mesenchymal transition of cancer cells and translation of Snail.
      HCCReaderYTHDF2DownEGFRYTHDF2 directly bound to the m6A modification site of EGFR 3′-UTR and promote the degradation of EGFR mRNA in HCC2019
      • Zhong L.
      • Liao D.
      • Zhang M.
      • Zeng C.
      • Li X.
      • Zhang R.
      • et al.
      YTHDF2 suppresses cell proliferation and growth via destabilizing the EGFR mRNA in hepatocellular carcinoma.
      HCC & TMReaderYTHDF2DownIL-11, SERPINE2YTHDF2 processes the decay of IL11 and SERPINE2 mRNA which are important for vessel normalization and inflammation2019
      • Hou J.
      • Zhang H.
      • Liu J.
      • Zhao Z.
      • Wang J.
      • Lu Z.
      • et al.
      YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.
      HCCReaderIGF2BP1UpC-MYC, MKI67IGF2BP1 stabilises the c-MYC and MKI67 mRNAs and increase their protein level in HCC2014
      • Gutschner T.
      • Hämmerle M.
      • Pazaitis N.
      • Bley N.
      • Fiskin E.
      • Uckelmann H.
      • et al.
      Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an important protumorigenic factor in hepatocellular carcinoma.
      HCCReaderIGF2BP1UpSRFIGF2BP1 enhances the expression of several SRF-target genes including PDLIM7 and FOXK1 and promotes tumour cell growth in vitro2018
      • Muller S.
      • Glass M.
      • Singh A.K.
      • Haase J.
      • Bley N.
      • Fuchs T.
      • et al.
      IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner.
      HCCReaderIGF2BP1GLI1LINC01093 can interact with IGF2BP1 and facilitate GLI1 mRNA decay in HCC cells2019
      • He J.
      • Zuo Q.
      • Hu B.
      • Jin H.
      • Wang C.
      • Cheng Z.
      • et al.
      A novel, liver-specific long noncoding RNA LINC01093 suppresses HCC progression by interaction with IGF2BP1 to facilitate decay of GLI1 mRNA.
      HCCReaderIGF2BP1lncRNA HULCIGF2BP1 recruits the CCR4-NOT complex and initiates the degradation of lncRNA HULC2013
      • Hämmerle M.
      • Gutschner T.
      • Uckelmann H.
      • Ozgur S.
      • Fiskin E.
      • Gross M.
      • et al.
      Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1).
      HCCReaderIGF2BP1/3LINC01138IGF2BP1/3 stabilise LINC01138 which is associated with poor outcomes of HCC patients2018
      • Li Z.
      • Zhang J.
      • Liu X.
      • Li S.
      • Wang Q.
      • Chen Di
      • et al.
      The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma.
      HCCEraserFTOUpPKM2FTO could induce tumorigenesis of HCC via regulating PKM2 at m6A modification-dependent manner2019
      • Li J.
      • Zhu L.
      • Shi Y.
      • Liu J.
      • Lin L.
      • Chen X.
      m6A demethylase FTO promotes hepatocellular carcinoma tumorigenesis via mediating PKM2 demethylation.
      ICCWriterWTAPUpWTAP is overexpressed in cholangiocarcinoma and regulates motility of cholangiocarcinoma cells2012
      • Jo H.J.
      • Shim H.E.
      • Han M.E.
      • Kim H.J.
      • Kim K.S.
      • Baek S.
      • et al.
      WTAP regulates migration and invasion of cholangiocarcinoma cells.
      ICCEraserFTODownTEAD2The mRNA stability of TEAD2 is increased by inhibiting FTO in ICC cell line2019
      • Rong Z.X.
      • Li Z.
      • He J.J.
      • Liu L.Y.
      • Ren X.X.
      • Gao J.
      • et al.
      Downregulation of fat mass and obesity associated (FTO) promotes the progression of intrahepatic cholangiocarcinoma.
      HBWriterMETTL3UpCTNNB1METTL3 promotes the proliferation of hepatoblastoma by regulating CTNNB12019
      • Liu L.
      • Wang J.
      • Sun G.
      • Wu Q.
      • Ma J.
      • Zhang X.
      • et al.
      m6A mRNA methylation regulates CTNNB1 to promote the proliferation of hepatoblastoma.
      TMWriterMETTL3HDGFMETTL3 stimulates the m6A modification of HDGF mRNA which enhances TM2019
      • Wang Q.
      • Chen C.
      • Ding Q.
      • Zhao Y.
      • Wang Z.
      • Chen J.
      • et al.
      METTL3-mediated m(6)A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance.
      TMWriterMETTL3miRNA1246METTL3 promotes CRC liver metastasis via METTL3/miR1246/SPRED2 axis2019
      • Peng W.
      • Li J.
      • Chen R.
      • Gu Q.
      • Yang P.
      • Qian W.
      • et al.
      Upregulated METTL3 promotes metastasis of colorectal cancer via miR-1246/SPRED2/MAPK signaling pathway.
      TMReaderIGF2BP2circNSUN2CircNSUN2 promotes liver metastasis of CRC through the HMGA2 pathway2019
      • Chen R.X.
      • Chen X.
      • Xia L.P.
      • Zhang J.X.
      • Pan Z.Z.
      • Ma X.D.
      • et al.
      N(6)-methyladenosine modification of circNSUN2 facilitates cytoplasmic export and stabilizes HMGA2 to promote colorectal liver metastasis.
      circRNA, circular RNA; CRC, colorectal carcinoma; EGFR, epidermal growth factor receptor; FOXK1, forkhead box K1; FTO, Fat mass and obesity-associated gene; HB, hepatoblastoma; HCC, hepatocellular carcinoma; HDGF, hepatoma-derived growth factor; HMGA2, high-mobility group AT-hook 2; ICC, intrahepatic cholangiocarcinoma; IGFBPs, insulin-like growth factor 2 mRNA binding proteins; IL11, interleukin 11; LINC, long intergenic non-coding RNA; LncRNA, long-noncoding RNA; m6A, N6-methyladenosine; miRNA, microRNA; NSUN2, NOP2/Sun RNA methyltransferase family member 2; PDLIM7, PDZ And LIM domain 7; PKM2, pyruvate kinase M2; RDM1, RAD52 motif containing 1; SOCS2, suppressor of cytokine signalling 2; SRF, serum response factor; TEAD2, TEA domain transcription factor 2; TM, tumor metastasis; UTR, untranslated region; WTAP, Wilms' tumour 1 associating protein; YTHDF2, YTH domain family protein 2.

      Therapeutic potential of targeting m6A regulators

      Since m6A RNA methylation play an important role in the progression of different liver diseases, manipulating specific m6A regulators may offer potential therapeutic approaches. FTO is one of the most well-studied m6A regulators and several inhibitors have been identified to target it. The natural product rhein was the first potent FTO inhibitor identified, displaying good inhibitory activity and increasing cellular m6A levels in vitro
      • Chen B.
      • Ye F.
      • Yu L.
      • Jia G.
      • Huang X.
      • Zhang X.
      • et al.
      Development of cell-active N6-methyladenosine RNA demethylase FTO inhibitor.
      ; however, it is not a very selective FTO inhibitor as it also targets ALKBH5, and its therapeutic efficacy in treating cancers has not yet been tested. Entacapone, an FDA-approved drug used to treat Parkinson's disease, has also been identified as a potential FTO inhibitor that may be useful for treating metabolic disorders.
      • Peng S.
      • Xiao W.
      • Ju D.
      • Sun B.
      • Hou N.
      • Liu Q.
      • et al.
      Identification of entacapone as a chemical inhibitor of FTO mediating metabolic regulation through FOXO1.
      Meclofenamic acid (MA) used to be a non-steroidal anti-inflammatory drug but is now regarded as a highly selective FTO inhibitor.
      • Huang Y.
      • Yan J.
      • Li Q.
      • Li J.
      • Gong S.
      • Zhou H.
      • et al.
      Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5.
      A recent study has demonstrated that MA can prevent oleic acid/dexamethasone-induced increases in total triglycerides in primary hepatocytes.
      • Hu Y.
      • Feng Y.
      • Zhang L.
      • Jia Y.
      • Cai D.
      • Qian S.B.
      • et al.
      GR-mediated FTO transactivation induces lipid accumulation in hepatocytes via demethylation of mA on lipogenic mRNAs.
      In addition, some natural products targeting RNA methylation have also been reported to have hepatoprotective effects. For instance, the methyl donor betaine can alleviate NAFLD in mice in an FTO-dependent manner,
      • Chen J.
      • Zhou X.
      • Wu W.
      • Wang X.
      • Wang Y.
      FTO-dependent function of N6-methyladenosine is involved in the hepatoprotective effects of betaine on adolescent mice.
      while the yellow polyphenolic pigment curcumin has been shown to attenuate LPS-induced liver injury and liver lipid metabolism disorder by increasing m6A RNA methylation
      • Lu N.
      • Li X.
      • Yu J.
      • Li Y.
      • Wang C.
      • Zhang L.
      • et al.
      Curcumin attenuates lipopolysaccharide-induced hepatic lipid metabolism disorder by modification of m6A RNA methylation in Piglets.
      ; however, further studies are required to investigate the side effects of these natural products.
      Numerous small molecule inhibitors have been identified to target m6A regulators, and hold therapeutic potential for treating various liver diseases.
      The effects of m6A modification inhibitors have been poorly studied in liver cancer. However, these inhibitors may provide potential therapies for other types of cancer, such as acute myeloid leukaemia (AML)
      • Su R.
      • Dong L.
      • Li C.
      • Nachtergaele S.
      • Wunderlich M.
      • Qing Y.
      • et al.
      R-2HG exhibits anti-tumor activity by targeting FTO/mA/MYC/CEBPA signaling.
      ,
      • Huang Y.
      • Su R.
      • Sheng Y.
      • Dong L.
      • Dong Z.
      • Xu H.
      • et al.
      Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia.
      and glioblastoma.
      • Cui Q.
      • Shi H.
      • Ye P.
      • Li L.
      • Qu Q.
      • Sun G.
      • et al.
      m6A RNA methylation regulates the Self-Renewal and tumorigenesis of glioblastoma stem cells.
      Treatment with the FTO inhibitor MA-2, an ethyl ester MA derivative, has been shown to inhibit glioblastoma stem cell (GSC) growth and GSC-induced tumorigenesis in vivo.
      • Cui Q.
      • Shi H.
      • Ye P.
      • Li L.
      • Qu Q.
      • Sun G.
      • et al.
      m6A RNA methylation regulates the Self-Renewal and tumorigenesis of glioblastoma stem cells.
      R-2HG, an oncometabolite produced by mutant IDH1/2 enzymes, has been reported to inhibit FTO activity and increase cellular m6A modification levels in AML while also exhibiting antitumor activity via FTO/m6A/MYC/CEBPA signalling.
      • Su R.
      • Dong L.
      • Li C.
      • Nachtergaele S.
      • Wunderlich M.
      • Qing Y.
      • et al.
      R-2HG exhibits anti-tumor activity by targeting FTO/mA/MYC/CEBPA signaling.
      Another compound, known as FB23-2, is also considered an effective FTO inhibitor and displays therapeutic effects in AML models.
      • Huang Y.
      • Su R.
      • Sheng Y.
      • Dong L.
      • Dong Z.
      • Xu H.
      • et al.
      Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia.
      Although there are no small molecule inhibitors yet targeting METTL3 and other m6A regulators that showed consistent upregulation in liver cancer, high-resolution crystal structures of METTL3, METTL14, and other m6A regulators have been already determined,
      • Wang P.
      • Doxtader K.A.
      • Nam Y.
      Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases.
      which may provide a basis for structure-guided drug design.
      • Boriack-Sjodin P.A.
      • Ribich S.
      • Copeland R.A.
      RNA-modifying proteins as anticancer drug targets.

      Discussion and perspectives

      A growing number of mRNA modifications have been identified due to advances in RNA sequencing; however, there is still an unmet biological need for new sequencing technologies. Scientists are inventing more sensitive sequencing methods that use less RNA input, which is very important for the research and clinical applications of epitranscriptomics. Currently, there is a lack of adequate methods for detecting multiple RNA modifications simultaneously within the same transcript, and hence, the relationship between m6A and other RNA modifications is largely unclear. Here, we highlight the essential effects of m6A modifications on liver disease phenotypes by affecting underlying RNA metabolism. HCC usually develops in livers with chronic diseases, such as viral hepatitis, fibrosis, and fatty liver disease.
      • Brown Z.J.
      • Heinrich B.
      • Greten T.F.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      To date, most studies of m6A in HCC have been carried out in vitro; thus, there is a lack of in vivo evidence and appropriate HCC mouse models are required to fully elucidate the regulatory networks of m6A modifications in liver cancer carcinogenesis. The m6A enzyme levels can be estimated by using liver biopsy tissues, thus some m6A regulators such as METTL3, METTL14, WTAP, and YTHDF2 could be potential prognostic markers for HCC,
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • et al.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N(6) -methyladenosine-dependent primary MicroRNA processing.
      ,
      • Chen M.
      • Wei L.
      • Law C.T.
      • Tsang F.H.
      • Shen J.
      • Cheng C.L.
      • et al.
      RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.
      ,
      • Chen Y.
      • Peng C.
      • Chen J.
      • Chen D.
      • Yang B.
      • He B.
      • et al.
      WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.
      METTL3 may also be a potential prognostic marker for HB.
      • Liu L.
      • Wang J.
      • Sun G.
      • Wu Q.
      • Ma J.
      • Zhang X.
      • et al.
      m6A mRNA methylation regulates CTNNB1 to promote the proliferation of hepatoblastoma.
      The current challenge for scientists is to study blood-based m6A related prognostic markers for liver cancer using extracellular microRNAs or circulating tumour cells.
      Future studies should use mouse models to investigate dynamic epitranscriptomic alterations during disease progression and the effect of m6A inhibitors on treating liver diseases.
      m6A modification is ubiquitous in mammalian cells and can be gained via SNPs, with several genome-wide association studies reporting the association between SNPs and hepatocarcinogenesis.
      • Nahon P.
      • Zucman-Rossi J.
      Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis.
      Unfortunately, the roles of m6A motif-related SNPs in HCC have not been well-studied, since these may change the fate of target mRNAs and lead to different consequences. In addition, since most liver diseases arise from multiple environmental factors, such as diet, alcohol, and xenobiotics, whether these environmental exposures can change liver epitranscriptomics is a major concern and may extend our understanding of the aetiology of liver diseases. Although the associations between m6A modifications and hepatic lipid metabolism, hepatitis virus infection, and liver cancer have been studied, little is known about the dynamic alteration and function of m6A modifications during liver fibrosis, autoimmune liver disease, and ischemia reperfusion injury. Therefore, epitranscriptomic information should be collected for each liver disease to fully evaluate the underlying mechanism and dynamic alteration in RNA modifications during liver disease progression.
      RNA modifications play a critical role in various liver diseases; however, little is known about their role in hepatic physiological processes. Thus, future studies should address many key aspects of the liver epitranscriptomic regulatory network under physiological conditions. For example, m6A modifications have been reported to control embryonic stem cell differentiation,
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      while the role of m6A modifications in hepatocyte or biliary epithelial cell differentiation remain unclear. Future efforts should also explore whether RNA modifications are involved in liver regeneration and whether liver metabolites affect RNA modifications in the liver, as the largest metabolic site in the human body.
      The discovery of novel m6A modification inhibitors has paved the way for m6A-based therapeutics; however, little is known about their effects in liver disease. The specificity and side effects of these drugs should be clearly clarified. Besides, it is important to determine whether these inhibitors influence other RNA modifications such as m1A and m6Am, since this could lead to uncertain consequences; thus, more studies on these inhibitors are required in vitro and in vivo. m6A modification is involved in various physiological processes, m6A writers and readers modulate almost every step of RNA metabolism; therefore, FTO, which plays an essential role in both adipogenesis and tumourigenesis, seems to be an ideal target. FTO inhibitors may modulate hepatic lipid metabolism in patients with NAFLD and may also protect hepatocytes from HCV infection. Mouse NAFLD or viral hepatitis models should be used to test the effects of m6A inhibitors on liver diseases. Besides, FTO is highly expressed in HCC and related to poor survival, thus FTO inhibitors such as rhein, entacapone, and MA might suppress the progression of HCC. More importantly, recent studies have demonstrated that FTO impairs IFNγ-induced killing of melanoma cells in vitro by upregulating PD-1, FTO knockdown sensitizes melanoma cells to anti-PD-1 treatment in mice.
      • Yang S.
      • Wei J.
      • Cui Y.H.
      • Park G.
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      In addition, combining anti-PD-L1 treatment with YTHDF1 depletion can improve the outcomes of tumour-bearing mice. Patients with colon cancer and lower expression of YTHDF1 in tumour stroma have also been shown to have more CD8+ T cell infiltration.
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      Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells.
      While it is unclear whether m6A modifications in liver cancer are also correlated with a T cell-infiltrated tumour microenvironment, targeting one specific m6A regulator may interfere with the antitumour immune responses in liver cancer development. Moreover, it is reported that pancreatic cancer cells that overexpress ALKBH5 are much more sensitive to anticancer reagents such as gemcitabine.
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      • Wang Y.
      • Wang Y.
      • Bi Y.
      • et al.
      m6A demethylase ALKBH5 inhibits pancreatic cancer tumorigenesis by decreasing WIF-1 RNA methylation and mediating Wnt signaling.
      FTO can regulate the chemoradiotherapy resistance of cervical squamous cell carcinoma by targeting β-catenin.
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      • et al.
      FTO regulates the chemo-radiotherapy resistance of cervical squamous cell carcinoma (CSCC) by targeting β-catenin through mRNA demethylation.
      Thus, combining appropriate m6A inhibitors with existing medical approaches, such as immune checkpoint blockade, chemotherapy, or radiotherapy, may lead to more effective therapies; however, further research is needed to confirm the use of RNA modifications as an effective treatment approach for liver diseases.

      Abbreviations

      ADAR, adenosine deaminase acting on RNA; ALKBH3/5, alkB homolog 3/5; ALYREF, Aly/REF export factor; AML, acute myeloid leukaemia; APOB, apolipoprotein B; CIDEC, cell death inducing DFFA like effector c; circRNA, circular RNA; CRC, colorectal carcinoma; CTSB/D/L, cathepsin B/D/L; CYP450, cytochrome P450; DC, dendritic cell; DDX46, DEAD-box helicase 46; EGFR, epidermal growth factor receptor; Ehhadh, enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase; eIF3h, eukaryotic translation initiation factor 3 subunit h; EIF4G2, eukaryotic translation initiation factor 4 gamma 2; EMT, epithelial-mesenchymal transition; FASN, fatty acid synthase; FOXK1, forkhead box K1; Foxo1, forkhead box O1; FTO, Fat mass and obesity-associated gene; HB, hepatoblastoma; HCC, hepatocellular carcinoma; HDGF, hepatoma-derived growth factor; hm5C, 5-hydroxymethylcytidine; HMGA2, high-mobility group AT-hook 2; ICC, intrahepatic cholangiocarcinoma; IDH1/2, isocitrate dehydrogenase 1/2; IGF2, insulin-like growth factor 2; IGFBP1/2/3, insulin-like growth factor 2 mRNA binding protein 1/2/3; IL11, interleukin 11; LINC, long intergenic non-coding RNA; LncRNA, long non-coding RNA; LPS, lipopolysaccharide; m1A, N1-methyladenosine; m5C, 5-methylcytosine; m6A, N6-methyladenosine; m6Am, N6,2′-O-dimethyladenosine; MA, meclofenamic acid; METTL3/14, methyltransferase-like 3/14; MGAT1, alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase; miRNA, microRNA; MTTP, microsomal triglyceride transfer protein; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NSUN2, NOP2/Sun RNA methyltransferase family member 2; PABP1, poly A binding protein 1; PAR-CLIP, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation; PCIF1, phosphorylated CTD interacting factor 1; PDLIM7, PDZ And LIM domain 7; PGC1α, PPARG coactivator 1 α; PKM2, pyruvate kinase M2; PPARα, peroxisome proliferator activated receptor-α; RBM, RNA-binding motifs; RDM1, RAD52 motif containing 1; SCD1, stearoyl-CoA desaturase-1; SIRT1, sirtuin 1; SNP, single nucleotide polymorphism; SOCS, suppressor of cytokine signalling; SREBP1, sterol regulatory element-binding protein 1; SRF, serum response factor; TIRAP, TIR domain containing adaptor protein; TM, tumour metastasis; UTR, untranslated region; WTAP, Wilms' tumour 1 associating protein; YBX1, Y-box binding protein 1; YTHDC1/2, YTH domain containing 1/2; YTHDF1/2/3, YTH domain family protein 1/2/3.

      Financial support

      This study was supported by National Key Research and Development Program of China ( 2017YFC0908102 to Q.X.), National Natural Science Foundation of China ( 81972205 & 81670598 to Q.X.) and Most Important Clinical Medical Center of Shanghai Health and Family Planning Commission ( 2017ZZ01018 to Q.X.).

      Authors' contributions

      Zhicong Zhao and Jiaxiang Meng drafted the manuscript. Rui Su and Jun Zhang provided feedback and guidance. Jianjun Chen, Xiong Ma and Qiang Xia revised the manuscript. All authors approved the final manuscript.

      Conflict of interest

      J.C. is the scientific founder of Genovel Biotech Corp. and holds equities with the company. Z.Z., J.M., R.S., J.Z., X.M. and Q.X. declare no conflicts of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

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