title: Hypomethylation of the hsa-miR-191 locus causes high expression of hsa-mir-191 and promotes the epithelial-to-mesenchymal transition in hepatocellular carcinoma.
abstract: hsa-miR-191 is highly expressed in hepatocellular carcinoma (HCC), but the factors regulating this elevated expression are unknown. This study aimed to investigate the epigenetic mechanisms of increased hsa-miR-191 expression by analyzing the relationship between the DNA methylation status of hsa-miR-191 and miR-191 expression. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing PCR, Northern blot, and quantitative real-time PCR were performed to examine hsa-miR-191 methylation and expression levels. Western blot, transwell, and scratch assays were performed to examine the function and molecular mechanisms of hsa-miR-191. Approximately 58.9% of hsa-miR-191 expression was higher in HCC tissues than in adjacent noncancerous tissues; this high expression was associated with poor prognosis. The hypomethylation observed in some HCC cell lines and HCC tissues was correlated with the hsa-miR-191 expression level. This correlation was validated by treatment with the 5-aza-DAC demethylation agent. The level of hypomethylation was 63.0% in 73 clinical HCC tissue samples and was associated with increased (2.1-fold) hsa-miR-191 expression. The elevated expression of hsa-miR-191 in the SMMC-771 HCC cell line induced the cells to transition into mesenchymal-like cells; they exhibited characteristics such as loss of adhesion, down-regulation of epithelial cell markers, up-regulation of mesenchymal cell markers, and increased cell migration and invasion. Inhibiting hsa-miR-191 expression in the SMMC-7721 cell line reversed this process (as assessed by cell morphology and cell markers). Furthermore, hsa-miR-191 probably exerted its function by directly targeting TIMP metallopeptidase inhibitor 3 and inhibiting TIMP3 protein expression. Our results suggest that hsa-miR-191 locus hypomethylation causes an increase in hsa-miR-191 expression in HCC clinical tissues and that this expression induces HCC cells to transition into mesenchymal-like cells.
authors.affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Cancer Institute, Shanghai, China.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: He
authors.forename: Ying
authors.initials: Y
authors.lastname: Cui
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Gu
authors.forename: Shicheng
authors.initials: S
authors.lastname: Guo
authors.forename: Kelong
authors.initials: K
authors.lastname: Ma
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Luo
mesh_terms: D001374:Azacitidine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D019175:DNA Methylation / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness; D011379:Prognosis; D012333:RNA, Messenger / Q000096:biosynthesis; D060888:Real-Time Polymerase Chain Reaction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000096:biosynthesis
chemical_list: C546177:MIRN191 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D019717:Tissue Inhibitor of Metalloproteinase-3; D001374:Azacitidine