Regulation and tumor-suppressive function of the miR-379/miR-656 (C14MC) cluster in cervical cancer.

ABSTRACT

Cervical cancer (CC) is a key contributor to cancer-related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR-379/miR-656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5-aza-2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation-regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral-based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post-transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer-related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT, MYC, and cyclin E1 (CCNE1) and the overexpression of p16, p21, and p27. We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca2+ levels, and lipid peroxidation rates, and inhibits epithelial-mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase-3 (PDK3) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR-494-mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor-suppressive and methylation-regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.