Up-regulation of microRNA 506 leads to decreased Cl/HCO3 anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis

Authors

  • Jesús M. Banales,

    Corresponding author
    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
    2. Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN
    3. Department of Gastroenterology, Donostia Hospital, Biodonostia Institute, CIBERehd, University of Basque Country, San Sebastián, Spain
    • Division of Gene Therapy and Hepatology, Center for Applied Medical Research, University of Navarra School of Medicine, Avenida Pío XII 55, E-31008 Pamplona, Spain
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    • fax: +34 948-194717

  • Elena Sáez,

    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
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  • Miriam Úriz,

    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
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  • Sarai Sarvide,

    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
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  • Aura D. Urribarri,

    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
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  • Patrick Splinter,

    1. Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN
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  • Pamela S. Tietz Bogert,

    1. Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN
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  • Luis Bujanda,

    1. Department of Gastroenterology, Donostia Hospital, Biodonostia Institute, CIBERehd, University of Basque Country, San Sebastián, Spain
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  • Jesús Prieto,

    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
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  • Juan F. Medina,

    1. Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and Center for Applied Medical Research, University of Navarra, and Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain
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  • Nicholas F. LaRusso

    1. Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN
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  • Potential conflict of interest: Nothing to report.

  • These authors contributed equally to this work and share senior authorship.

Abstract

Cl/HCOmath image anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) — predicted as candidate to target AE2 mRNA — for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. Conclusion: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease. (HEPATOLOGY 2012)

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