The Tumor-Suppressive miR-497-195 Cluster Targets Multiple Cell-Cycle Regulators in Hepatocellular Carcinoma

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and commonly deregulated carcinogenesis. To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression followed by pathway analysis, revealed a significant enrichment of cell cycle regulators. Among the candidates, we successfully identified CCNE1, CDC25A, CCND3, CDK4, and BTRC as direct targets for miR-497 and rniR-195. Moreover, target genes frequently upregulated in HCC in a tumor-specific manner, such as CDK6, CCNE1, CDC25A and CDK4, showed an inverse correlation in the expression of miR-195 and miR-497, and their targets. These results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to the aberrant cell proliferation in hepatocarcinogenesis.