Highlights
- •CCA tissues are characterized by miR upregulation, increased miR biogenesis pathway expression and miR heterogeneity.
- •Most miRs upregulated in CCA resulted in increased proliferation when introduced into human cholangiocyte models in vitro.
- •MiR-27a-3p affects FoxO signalling in individuals with CCA in vitro and in vivo.
- •CRISPR/Cas9 nickase knockout of miR-27a abrogates tumorigenicity in vitro and in vivo.
Background & Aims
Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins
of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways,
functioning as potent epigenetic regulators of transcriptional output. We aimed to
characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis
and cell behaviour.
Methods
Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues,
and 22 normal livers. High-throughput miR mimic screens were performed in three primary
human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together
with miR screening data identified an oncogenic miR for characterization. MiR-mRNA
interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were
generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts.
Results
In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA
and surrounding liver tissues, including 135 that were upregulated in tumours. CCA
tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway
expression. Unsupervised hierarchical clustering of tumour miRNomes identified three
subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs
that consistently increased proliferation of three primary cholangiocyte models and
were upregulated in CCA tissues regardless of anatomical location, among which only
miR-27a-3p had consistently increased expression and activity in several cohorts.
FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through
targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth.
Conclusions
The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis
in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA.
Impact and implications
Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic
and non-genetic alterations, but the functional roles of these non-genetic events
remain poorly understood. By unveiling global miRNA upregulation in patient tumours
and their functional ability to increase proliferation of cholangiocytes, these small
non-coding RNAs are implicated as critical non-genetic alterations promoting biliary
tumour initiation. These findings identify possible mechanisms for transcriptome rewiring
during transformation, with potential implications for patient stratification.
Graphical abstract

Graphical Abstract
Keywords
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References
- Cholangiocarcinoma 2020: the next horizon in mechanisms and management.Nat Rev Gastroenterol Hepatol. 2020; 17: 557-588
- Cholangiocarcinoma landscape in Europe: diagnostic, prognostic and therapeutic insights from the ENSCCA Registry.J Hepatol. 2022 May; 76: 1109-1121
- Desmoplastic tumor microenvironment and immunotherapy in cholangiocarcinoma.Trends Cancer. 2018; 4: 239-255
- Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis.Science. 2021; 373eabj0486
- Genomic spectra of biliary tract cancer.Nat Genet. 2015; 47: 1003-1010
- Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations.J Hepatol. 2018; 68: 959-969
- Genomic perturbations reveal distinct regulatory networks in intrahepatic cholangiocarcinoma.Hepatology. 2018; 68: 949-963
- Pan-cancer analysis of whole genomes.Nature. 2020; 578: 82-93
- Whole-genome and epigenomic landscapes of etiologically distinct subtypes of cholangiocarcinoma.Cancer Discov. 2017; 7: 1116-1135
- Epigenome remodeling in cholangiocarcinoma.Trends Cancer. 2019; 5: 335-350
- Transcriptomic profiling reveals hepatic stem-like gene signatures and interplay of miR-200c and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma.Hepatology. 2012; 56: 1792-1803
- Noncoding RNA in cholangiocarcinoma.Semin Liver Dis. 2019; 39: 13-25
- miR-21 and KLF4 jointly augment epithelialmesenchymal transition via the Akt/ERK1/2 pathway.Int J Oncol. 2017; 50: 1109-1115
- MiR-21 promotes intrahepatic cholangiocarcinoma proliferation and growth in vitro and in vivo by targeting PTPN14 and PTEN.Oncotarget. 2015; 6: 5932-5946
- MIR21 drives resistance to heat shock protein 90 inhibition in cholangiocarcinoma.Gastroenterology. 2018; 154: 1066-10679 e5
- Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.Gastroenterology. 2012; 142: 1021-1031 e15
- Application of patient-derived liver cancer cells for phenotypic characterization and therapeutic target identification.Int J Cancer. 2019; 144: 2782-2794
- Ursodeoxycholic acid inhibits hepatic cystogenesis in experimental models of polycystic liver disease.J Hepatol. 2015; 63: 952-961
- SOX17 regulates cholangiocyte differentiation and acts as a tumor suppressor in cholangiocarcinoma.J Hepatol. 2017; 67: 72-83
- Identification of a pan-gamma-secretase inhibitor response signature for notch-driven cholangiocarcinoma.Hepatology. 2020; 71: 196-213
- Integrative genomic analysis of cholangiocarcinoma identifies distinct IDH-mutant molecular profiles.Cell Rep. 2017; 19: 2878-2880
- miRNA activity inferred from single cell mRNA expression.Sci Rep. 2021; 11: 9170
- The regulation of FOXO1 and its role in disease progression.Life Sci. 2018; 193: 124-131
- miRNA profiling of biliary intraepithelial neoplasia reveals stepwise tumorigenesis in distal cholangiocarcinoma via the miR-451a/ATF2 axis.J Pathol. 2020; 252: 239-251
- Pan-cancer characterisation of microRNA across cancer hallmarks reveals microRNA-mediated downregulation of tumour suppressors.Nat Commun. 2018; 9: 5228
- Most mammalian mRNAs are conserved targets of microRNAs.Genome Res. 2009; 19: 92-105
- Elucidating MicroRNA regulatory networks using transcriptional, post-transcriptional, and histone modification measurements.Cell Rep. 2016; 14: 310-319
- Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis.Gut. 2022 Feb; 71: 391-401
- Dysregulated miR-124 and miR-200 expression contribute to cholangiocyte proliferation in the cholestatic liver by targeting IL-6/STAT3 signalling.J Hepatol. 2015; 62: 889-896
- Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells.Hepatology. 2005; 42: 1329-1338
- Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression.Cancer Res. 2006; 66: 10517-10524
- MicroRNA-506 promotes primary biliary cholangitis-like features in cholangiocytes and immune activation.Hepatology. 2018; 67: 1420-1440
- miR-425 regulates inflammatory cytokine production in CD4(+) T cells via N-Ras upregulation in primary biliary cholangitis.J Hepatol. 2017; 66: 1223-1230
- Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma.J Hepatol. 2020; 73: 315-327
- Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes.Gastroenterology. 2013; 144: 829-840
- FoxOs at the crossroads of cellular metabolism, differentiation, and transformation.Cell. 2004; 117: 421-426
- FoxO3 inactivation promotes human cholangiocarcinoma tumorigenesis and chemoresistance through Keap1-Nrf2 signaling.Hepatology. 2016; 63: 1914-1927
- MicroRNA-1269 promotes proliferation in human hepatocellular carcinoma via downregulation of FOXO1.BMC Cancer. 2014; 14: 909
- Downregulation of microRNA-196a inhibits human liver cancer cell proliferation and invasion by targeting FOXO1.Oncol Rep. 2017; 38: 2148-2154
- FOXO1 couples metabolic activity and growth state in the vascular endothelium.Nature. 2016; 529: 216-220
- Multi-omics analysis identifies FoxO1 as a regulator of macrophage function through metabolic reprogramming.Cell Death Dis. 2020; 11: 800
- Therapeutic advances of miRNAs: a preclinical and clinical update.J Adv Res. 2021; 28: 127-138
Article info
Publication history
Published online: October 27, 2022
Accepted:
October 10,
2022
Received in revised form:
October 7,
2022
Received:
March 29,
2022
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