DNMT1 facilitates proliferation and metastasis of breast cancer by inducing MEG3 promoter methylation in MEG3/miR-494-3p/OTUD4 regulatory axis

Abstract Background: To explore the mechanism by which DNMT1 potentiates proliferation and metastasis of breast cancer by inducing MEG3 promoter methylation in MEG3/miR-494-3p/OTUD4 regulatory axis.Methods: Human breast cancer cell lines (MCF-7, MDA-MB-231, SKBR3) and human breast epithelial cell line MCF10A were selected for the experiments. The expression levels of DNMT1, MEG3, miR-494-3p and OTUD4 were detected by qRT-PCR. Western blot was used to detect the protein expression levels of DNMT1 and OTUD4. ChIP assay was adopted to verify the binding relationship between DNMT1 and MEG3 promoter region. MethPrimer software was applied to identify MEG3 promoter methylation while methylation-specific PCR was conducted to examine its methylation level. The targeted binding sites of miR-494-3p on MEG3/OTUD4 were predicted by bioinformatics and further verified by RNA binding protein immunoprecipitation assay plus dual-luciferase reporter gene assay. Cell proliferative, migratory and invasive abilities were measured by CCK-8, wound healing and Transwell assays.Results: DNMT1 was highly expressed while MEG3 was poorly expressed in breast cancer cells. Silencing DNMT1 inhibited the proliferation, migration and invasion of breast cancer cells by increasing gene expression of MEG3 through demethylation. MEG3 was identified as a ceRNA that regulated miR-494-3p expression via RNA sponging in breast cancer. In addition, miR-494-3p could bind to the 3’-UTR of OTUD4 mRNA, thus negatively regulating OTUD4 expression. A regulator axis formed by MEG3/miR-494-3p/OTUD4 was thus established and identified to have an impact on proliferation, invasion and migration of breast cancer. Moreover, overexpression of MEG3 could suppress tumor growth of breast cancer in vivo.Conclusion: Silencing DNMT1 induced demethylation of MEG3 promoter to promote MEG3 gene expression, in turn inhibiting the expression of miR-494-3p to elevate the expression of its downstream target OTUD4, thus weakening the proliferative, migratory and invasive abilities of breast cancer cells.