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

Abstract

Background To explore the mechanism of DNMT1 facilitating proliferation and metastasis of breast cancer cells by promoting 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 expressions of DNMT1, MEG3, miR-494-3p and OTUD4 were detected by qRT-PCR. Western blot was used to detect the protein expressions of DNMT1 and OTUD4. ChIP assay verified the binding relationship of DNMT1 and MEG3 promoter region. MEG3 methylation and its level were test by MethPrimer software and MSP, respectively. The targeted binding sites of miR-494-3p and MEG3/OTUD4 were predicted by bioinformatics. RIP and dual-luciferase reporter gene assays verified the combination of miR-494-3p and MEG3/OTUD4. Cell proliferation, migration and invasion abilities were detected 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 promoting the expression of lncRNA MEG3 through demethylation. MEG3 as a ceRNA regulated the expression of miR-494-3p in breast cancer. In addition, miR-494-3p could bind to the 3’-UTR of OTUD4, thus negatively regulating the expression of OTUD4 and forming the MEG3/miR-494-3p/OTUD4 regulatory axis that affected the proliferation, invasion and migration of breast cancer. Overexpression of MEG3 could inhibit breast cancer tumor growth in vivo. Conclusion Silencing DNMT1 promoted the expression of MEG3 through demethylation, which inhibited the expression of miR-494-3p to promote the expression of downstream target OTUD4, thus inhibiting the proliferation, migration and invasion abilities of breast cancer cells.