DNA methylation regulated microRNAs in human cervical cancer

Regulation of miRNA gene expression by DNA promoter methylation may represent a key mechanism to drive cervical cancer progression. In order to understand the impact of DNA promoter methylation on miRNAs at various stages of cervical carcinogenesis, we performed DNA methylation microarray on Normal Cervical Epithelium (NCE), Cervical Intraepithelial Neoplasia (CIN I-III) and Squamous Cell Carcinoma (SCC) tissues to identify differentially methylated miRNAs followed by validation by bisulfite sequencing. Further, expression of miRNAs was analyzed by qRT-PCR in clinical tissues and cervical cancer cell lines. Transcriptional activity was determined by luciferase assay. We identified a total of 69 hypermethylated and hypomethylated miRNA promoters encompassing 78 CpG islands in all except Y chromosome, among the three groups. The candidate DNA promoters of miR-424 were significantly hypermethylated and miR-200b and miR-34c were significantly hypomethylated in SCC compared to NCE (P<0.05). Expression of miR-424, miR-200b, and miR-34c were inversely correlated with promoter DNA methylation in tissue samples. Treatment of cell lines with 5-aza-2-deoxycytidine showed differential expression in all three miRNAs. We observed a decrease in miRNA promoter activity following in vitro SssI methylase treatment of miR-424, miR-200b, and miR-34c. Luciferase assay demonstrated that miR-200b and miR-424 functionally interacts with 3-UTR of HIPK3 and RBBP6 respectively and decreased their activity in presence of miR-200b and miR-424 mimics transfected in SiHa cells. Taken together, we have identified deregulation of miRNAs by aberrant DNA promoter methylation, leading to its transcriptional silencing during cervical carcinogenesis, which can be potential targets for diagnosis and therapy.