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MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells

  • Author Footnotes
    † Equal author contribution
    Lea Duwe
    Footnotes
    † Equal author contribution
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Author Footnotes
    † Equal author contribution
    Patricia Munoz-Garrido
    Footnotes
    † Equal author contribution
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Monika Lewinska
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Juan Lafuente-Barquero
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Letizia Satriano
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Dan Høgdall
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark

    Department of Oncology, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark
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  • Andrzej Taranta
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Boye S. Nielsen
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    Bioneer, Kogle Alle 2, DK-2970, Hørsholm, Denmark
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  • Awaisa Ghazal
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Matthias S. Matter
    Affiliations
    Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland
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  • Jesus M. Banales
    Affiliations
    Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain

    National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, “Instituto de Salud Carlos III”), Spain

    IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

    Department of Biochemistry and Genetics, School of Sciences, University of Navarra, Pamplona, Spain
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  • Blanca I. Aldana
    Affiliations
    Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
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  • Yu-Tang Gao
    Affiliations
    Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China
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  • Jens U. Marquardt
    Affiliations
    Department of Medicine I, University Medical Center Schleswig-Holstein-Campus Lübeck, 23558 Lübeck, Germany
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  • Lewis R. Roberts
    Affiliations
    Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA
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  • Rui C. Oliveira
    Affiliations
    Coimbra Institute for Clinical and Biomedical Research (iCBR) Area of Environment, Genetics and Oncobiology (CIMAGO), Institute of Biophysics, Faculty of Medicine, University of Coimbra, Portugal

    Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal
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  • Jill Koshiol
    Affiliations
    Division of Cancer Epidemiology and Genetics, NIH, USA
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  • Colm J. O'Rourke
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Jesper B. Andersen
    Correspondence
    Corresponding authors. Address: Biotech Research and Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Denmark; Tel.: (+45) 35325834.
    Affiliations
    Biotech Research & Innovation Centre (BRIC), Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen N, Denmark
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  • Author Footnotes
    † Equal author contribution
Open AccessPublished:October 27, 2022DOI:https://doi.org/10.1016/j.jhep.2022.10.012

      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

      Keywords

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