Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6-targeting miRNAs, miR-433 and miR-22, are down-regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin-5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin-5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin-5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. Conclusion: These data suggest that down-regulated Exportin-5 impairs the nuclear export of miR-433 and miR-22 precursor forms, causing a decrease in levels of mature miR-433 and miR-22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).
Bibliographical noteFunding Information:
Received September 18, 2017; accepted January 25, 2018. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.29832/suppinfo. Supported by National Institutes of Health grant R01CA183764 (to S.A.G.), The Randy Shaver Cancer Research and Community Fund Award (to S.A.G.), and The Hormel Foundation, grants P30DK084567 and R01DK24031 (to N.F.L.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The miRNA qPCR study in human samples were supported by Spanish Ministry of Economy and Competitiveness (J.M.B. [FIS PI15/01132 and Miguel Servet Program CON14/00129] and M.J.P. [FIS PI14/00399] cofinanced by “Fondo Europeo de Desarrollo Regional” [FEDER]), and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to J.M.B). These authors made an equal contribution. Copyright VC 2018 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.29832
© 2018 by the American Association for the Study of Liver Diseases.
Copyright 2019 Elsevier B.V., All rights reserved.
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't