Methylation-mediated regulation of tumor-suppressor function of the miR-379/656 (C14MC) cluster and its clinical utility in hepatocellular carcinoma

Hepatocellular carcinoma is a leading cause of cancer-related mortality. Several microRNAs play key roles in HCC development and progression. Epigenetic processes, such as DNA methylation, might regulate these RNAs during HCC pathogenesis. In this study, we show that the miR-379/656 cluster/C14MC acts as a tumor suppressor cluster and is epigenetically regulated by DNA methylation.

C14MC miRNA expression was determined in HCC cell lines using the nCounter assay and from the TCGA-LIHC clinical cohort. C14MC putative promoter was identified, characterized using cloning and luciferase assay, and the methylation status of promoter-bound CpGs was determined using bisulfite Sanger sequencing. The expressions of C14MC targets were experimentally validated by transcriptomic sequencing or transfecting mimics, followed by qRT-PCR. Furthermore, the diagnostic and prognostic significance of C14MC and its target interactome in HCC was assessed using clinical data from the TCGA-LIHC cohort.

C14MC was downregulated in HCC cell lines and in TCGA-LIHC. The loss of C14MC tumor suppressor function was directly regulated by the hypermethylation of promoter-CpGs. Reactivating specific C14MC miRNAs, such as miR-299-5p and miR-376c-3p via mimics, abrogated the expression of several target oncogenes, including PARP1, SPP1, RAD21, and CENPA, which regulate critical molecular pathways such as the p53 signaling and NF-κB signaling pathways in HCC. Additionally, overexpressing these miRNAs inhibited HCC cell migration and invasion. Also, C14MC and its target interactome exhibited significant clinical correlation in terms of survival outcomes of HCC patients.

This is the first study to show that C14MC is a methylation-dependent cluster in HCC. Several of these miRNAs and their targets can be used for early HCC diagnosis and prognosis. Thus, targeting C14MC can be useful in HCC management.