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

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. Several microRNAs (miRNAs) play key roles in HCC development and progression. The role of epigenetic processes like DNA methylation in the regulation of miRNAs is critical to 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. We demonstrated that C14MC is downregulated in HCC cell lines through nCounter assay and in clinical samples from The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma tissues. The C14MC promoter was identified and characterized through cloning and dual luciferase assay. Furthermore, we demonstrated that the loss of C14MC tumor suppressor function is directly regulated by the hypermethylation of promoter-bound CpGs, as shown in artificial methylation experiments. The reactivation of specific C14MC miRNAs, like miR-299-5p and miR-376c-3p via mimics, abrogated the expression of several target oncogenes, including PARP1, SPP1, RAD21, and CENPA that regulate critical molecular pathways such as the p53 signaling and NF-kappa B signaling pathways in HCC. Additionally, overexpression of miR-299-5p and miR-376c-3p inhibited HCC cell migration and invasion, suggesting that overexpression of candidate C14MC miRNAs can mitigate cancer hallmarks in HCC cells. Additionally, we have performed whole transcriptomic sequencing for RNA isolated from HCC spheroids generated from HepG2 cells to identify the DEGs regulated by C14MC. Furthermore, we checked for the clinical correlation of C14MC targets and their target genes in terms of survival outcomes and identified the key genes associated with prognostic potential in HCC. We conclude from our experimental findings that C14MC is a tumor-suppressor miRNA cluster and is regulated epigenetically by methylation 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.