DNA methylation regulates glioma cell cycle through down-regulating MiR-133a-5p expression

Abstract Background: MiRNAs plays a key role in regulating gene expression networks of various biological processes in many cancers. Results: Here, we analyzed miRNA expression profiles by miRNA microarray and verified by RT-PCR. It was shown that the expression difference of miR-133a-5p was most significantly and consistently downregulated. The proliferative capacity and cell cycle profile of cells transfected with miR-133a-5p mimic were assessed by colony forming assay and PI staining, respectively. The target gene of miR-133a-5p was predicted using TargetScan and verified by dual luciferase gene reporter assay. Western blotting and RT-PCR were used to analyze the expression levels of relevant factors. Methylation-specific quantitative PCR (MSP) was used to detect miR-133a-5p methylation levels. Epigenetic regulation of miR-133a-5p was assessed by treating the cells with the DNA methyltransferase inhibitor AZA or the histone deacetylase inhibitor TSA. We found that overexpression of miR-133a-5p inhibited cell proliferation, induced a cell cycle arrest and downregulated the expression of Cyclin D1, Cyclin D2, and cyclin dependent kinase 4 (CdK4). Peroxisome proliferator-activated receptor γ coactivator 1 alpha (PPARGC1A or PGC-1α) was verified as a target gene of miR-133a-5p. PGC-1α protein levels were significantly decreased in glioma cells following miR-133a-5p overexpression. Furthermore, forced expression of PGC-1α partly abrogated the anti-proliferative effects of miR-133a-5p. miR-133a-5p was hypermethylated in glioma cells, and AZA treatment significantly up-regulated its levels. Conclusions: MiR-133a-5p is downregulated in glioma cells through promoter hypermethylation, and its forced expression inhibits glioma cell proliferation and induces G1 phase arrest by targeting PGC-1α.