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Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, ChinaDepartment of Systems Biology, Beckman Research Institute of City of Hope, Monrovia, CA 91016, USA
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
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.
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
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.
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.
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.
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.
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,
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.
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.
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,
). 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,
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,
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.
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.
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.
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.
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.
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,
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
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.
; 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.
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.
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,
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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,
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.
(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.
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.
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.
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.
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.
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.
; 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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,
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,
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
; 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.
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.
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.
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.
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.
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
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
; 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.
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,
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)
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.
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,
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.
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,
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,
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.
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.
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.
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.
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.).
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.