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

Abstract

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and commonly deregulated in 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 miR-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.

Hypoxia‐induced miR‐497 decreases glioma cell sensitivity to TMZ by inhibiting apoptosis

“…Previous reports show that miR-497 is downregulated in hepatocellular carcinomas and breast cancers, functioning as a tumor suppressive miRNA [10]. However, our experimental data, along with our analysis of a published dataset, demonstrate that miR-497 is overexpressed in glioma.…”

“…Forced overexpression of miR-497 has been shown to inhibit breast cancer cell growth and invasion [9]. In hepatocellular carcinoma, miR-497 was reported to be a tumor suppressive miRNA, showing growthsuppressive activities by targeting CDK6, CCNE1, CDC25A, and CDK4 [10].…”

MicroRNA-497 regulates cell proliferation in hepatocellular carcinoma

“…These results indicate that miR-497 regulates PI3K/Akt pathway activation, consistently with results from a previous study on human colorectal cancer (12). Notably, both miRNA-497 and p53 cluster at 17p13.1 (21), and wild-type p53 inhibits IGF-1R expression, whereas mutant p53 increases IGF-1R expression (48,49). These findings suggest that abnormal transcription or translation of the chromosome 17p13.1 fragment may play a crucial role in hepatocarcinogenesis.…”

“…The results confirmed that the expression levels of miRNA-497 are decreased in HCC tumor tissues or HCC-derived cell lines compared with adjacent non-cancerous tissues or normal human L02 hepatocytes. Previously, Furuta et al (21) reported that miR-497 suppressed cell growth by targeting multiple cell-cycle regulators in HCC (21). These results were consistent with those of the current in vitro experiments in which exogenous overexpression of miRNA-497 was observed to inhibit MHCC-97H colony formation and tumor growth.…”

“…Recently, an increasing number of studies have revealed that microRNA-497 (miR-497) levels are decreased in tumors, and that it functions as a tumor suppressor in a number of types of human cancer, including colorectal, gastric, cervical and breast cancers, adrenocortical carcinoma and melanoma (12)(13)(14)(15)(16)(17)(18)(19)(20). Similarly, a study by Furuta et al (21) indicated that miR-497 targets multiple cell cycle regulators and suppresses cell cycle progression in vitro. However, whether miR-497 regulates other target genes in HCC is unknown.…”