MiR-93-5p Inhibits The EMT Of Breast Cancer Cells Via Targeting MKL1 And STAT3

Objective:Breast cancer is the most common malignancy in women,and metastasis and recurrence are the leading causes of death in breast cancer patients.Tumor metastasis is a multistep,multi-stage,multi-pathway,involving a series of complex processes of multiple gene changes.There is growing evidence that epithelial mesenchymal transition(EMT)is closely related to tumor metastasis.Revealing the molecular regulation mechanism of EMT and its relationship with the metastasis of breast cancer,for the prevention and treatment of breast cancer is of great significance.Methods:The pcDNA3.1-MRTFA-myc was contained a cDNA encoding amino acids1-931 of human MKL1,the pcDNA3.1-STAT3-flag was contained a cDNA amino acids 1-769 of human STAT3.The vector pcDNA3.1 alone was used as a negative.MKL1 and STAT3 were overexpressed in MCF-7 cells for wound-healing assays and transwell.To further determine whether MKL1-mediated transactivation of Vimentin promoter is dependent on the CAr G box,luciferase reporter vectors of Vimentin gene promoter were constructed:(1)WT-Vimentin-luc;(2)M-Vimentin-luc;(3)D-Vimentin-luc.To further confirm the specific binding of MKL1 to Vimentin promoter,Ch IP assays were performed in MCF-7 cells transfected with MKL1,MKL1/STAT3 or vector.In order to determine the potential role of mi R-93-5p in regulating breast cancer cell EMT to affect the migration of breast cancer cells,MCF-7 cells or MDA-MB-231 cells were transfected mi R-93-5p mimics or negative control(NC)or mi R-93-5p inhibitors or negative control(NC).To confirm whether Mi R-93-5p regulates the protein expression of MKL1 and STAT3 depends on its3’UTR region,following plasmids were constructed:(1)pmir GLO-MKL1-WT-3’UTR;(2)pmir GLO-STAT3-WT-3’UTR;(3)pmir GLO-MKL1-mut-3’UTR;(4)pmir GLO-STAT3-mut-3’UTR.Results:According to the above experimental program,we concluded:MKL1 and STAT3 synergistically promoted the MET of MCF-7 cells.MKL1 and STAT3 synergistically promotes the EMT marker(Vimentin)via its promoter CAr G box.Mi R-93-5p promotes EMT of human breast cancer cells.Mi R-93-5p silences the expression ofMKL1 and STAT3 via targeted their 3’UTR.The model of mi R-93-5p/MKL1/STAT3 to regulate the EMT to affect breast cancer migration.Conclusion:EMT is played an important role in tumor metastasis.MKL1 and STAT3 are involved in cell metabolism,survival and growth control.Previous studies have shown that MKL1 and STAT3 synergistically promote the migration of breast cancer cells.We are demonstrated that MKL1 and STAT3 affect the migration of breast cancer cells during mi RNA-mediated cell EMT.The expression of EMT marker protein Vimentin are promoted via MKL1 and STAT3 by binding to CAr G-box on its promoter.Our data showed that mi R-93-5p silenced the expression of MKL1 and STAT3 via targeted their 3’UTR.Therefore,mi R-93-5p mimics may be provided a means to reverse the EMT phenotypes and prevent cancer invasion and metastasis by targeting MKL1 and STAT3.