MRTF-A And STAT3 Synergistically Promote Breast Cancer Cell Migration Through Inhibition Of BRMS1

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis.Metastasis is a complex biological process,which associated with a variety of signaling pathways and transcription factors.Epigenetic modification,especially DNA methylation,is closely correlated with the expression of these genes.BRMS1 is a breast cancer metastasis suppressor gene that can inhibit the invasion and metastasis of tumor cells,but the mechanism by which BRMS1 is silenced and its relationship with MRTF-A and STAT3 is not fully clear.Thus,this study mainly focused on the roles of MRTF-A and STAT3 in BRMS1 epigenetic silencing and their effects on the migration of breast cancer cells.First,we verified the expression of BRMS1 in MCF-7 cells and MDA-MB-231 cells.The results showed that BRMS1 was less expressed in MDA-MB-231 cells.Furthermore,we used wound healing assay and transwell experiment to investigate the role of BRMS1 in breast cancer cell migration,and the results proved that the BRMS1 can inhibit the migration of breast cancer cells.The expression of migration markers(uPA and OPN)were detected by RT-PCR and Western Blot.The results demonstrated that BRMS1 could down-regulate the expression of migration markers.To determine whether the less expression of BRMS1 is related with the epigenetic modification,the BRMS1 promoter methylation status was examined by sodium bisulfite sequence analysis.The results showed that the BRMS1 promoter CpG island hypermethylation causes BRMS1 gene silence in MDA-MB-231 cells;the specific methyltransferase inhibitor 5-aza-dc treatment induced the BRMS1 re-expression in breast cancer MDA-MB-231 cells.Secondly,we confirmed the molecular mechanisms of MRTF-A and STAT3 synergistically promoting breast cancer cells migration by BRMS1 epigenetic modification.Our datas showed that overexpression of MRTF-A and STAT3 synergistically enhanced the expression of uPA and OPN,and inhibited the expression of BRMS1.The results demonstrated that overexpression of MRTF-A and STAT3 can cause BRMS1 silence as a result of enhancing the BRMS1 promoter methylation.The study also found that forced expression of MRTF-A and STAT3 enhanced the expression of DNMT1 in breast cancer cells.Then sodium bisulfite sequence analysis was performed to examine the BRMS1 promoter methylation status.Moreover,the luciferase assays illustrated that MRTF-A and STAT3 synergistically activated the transcription of the DNMT1 gene.Further analysis with site-directed mutagenesis and ChIP demonstrated that STAT3 binding site within the DNMT1 promoter region played key roles in the transactivation of DNMT1 by STAT3.Thus,this study provides important and novel insights into the roles of MRTF-A and STAT3 in regulating breast cell migration via inhibiting BRMS1 through epigenetic modification.This will provide the theoretical basis for the prevention and treatment and the prognosis of breast cancer.