Hypomethylation Causes MIR21 Overexpression in Tumors.

miR-21 is an oncogenic microRNA (miRNA) that is upregulated in many solid tumors. However, the effect of MIR21 hypomethylation on miR-21 expression in tumors and the mechanism of miR-21 DNA demethylation remain unclear. In this study, we confirmed that the expression of miR-21 was significantly increased in multiple tumors. We analyzed eight types of cancer, including breast cancer (BRCA), lung adenocarcinoma (LUAD), renal and renal clear cell carcinoma (KIRC), bladder urothelial carcinoma (BLCA), hepatocellular carcinoma (LIHC), lung squamous cell cancer (LUSC), renal papillary cell carcinoma (KIRP), and pancreatic adenocarcinoma (PAAD). MIR21 DNA methylation levels were elevated in these cancers. CpG loci located approximately 200 bp upstream of the transcription initiation site strongly affect MIR21 expression. We also confirmed MIR21 hypomethylation by pyrosequencing of fresh clear cell renal cell carcinoma (ccRCC) samples. Demethylating agent was proved to increase hsa-miR-21-5p level in HEK293T cells, while knockdown of DNA demethylases TET3 and TDG decreased MIR21 expression. In addition, we showed that the cg02515217 CpG locus in MIR21 promoter was a conserved binding site of transcription factors CEBPB, MEIS3, and TEAD4, which were co-expressed with miR-21 in tumors. These observations identified that gene hypomethylation regulated the expression of MIR21 in tumors.