MiRNA-429 Regulates Proliferation And Migration Of Breast Cancer Cells By Regulating EIF4E

Research purposes: The role of miRNA and its regulatory target genes as tumor markers in tumor occurrence and development is currently a research hotspot.Micro RNA-429(miRNA-429)is down regulated in breast cancer and plays an anti-tumor role.In breast cancer,the specific mechanism of its downstream target gene regulation is still unclear,which needs further experimental verification.Bioinformatics predicts that eukaryotic cell translation initiation factor 4E may be a potential downstream target gene of miRNA-429.Eukaryotic cell translation initiation factor 4E(e IF4E)has a regulatory role in breast cancer,and its expression level may be closely related to the occurrence,progress and prognosis of breast cancer.The purpose of this study is to explore the effects of eIF4E and miRNA-429 on the proliferation and migration of breast cancer cells,and predict and verify the binding targets of miRNA-429 and its target gene eIF4E.Through experiments,it is confirmed that miRNA-429 can regulate eIF4E by targeting it,so as to discuss the molecular mechanism of miRNA-429 in malignant behaviors such as proliferation and migration of breast cancer cells.Methods:1)Pathology confirmed that there were 20 pairs of invasive breast cancer tissues and corresponding paracancerous tissues from the Breast Surgery Department of the Affiliated Hospital of Qingdao University.The relative expression of eIF4E m RNA in breast cancer tissues and corresponding adjacent tissues was measured by real-time quantitative reverse transcription PCR(RT-q PCR).2)The relative expression of eIF4E m RNA and protein in breast cancer cells(MDA-MB-231,MDA-MB-468,MCF-7 and BT-549)and normal breast epithelial cells(MCF-10A)were detected by RT q PCR and Western blot.3)After cell transfection(si-e IF4E),RT-q PCR was used to detect the relative expression of eIF4E m RNA in breast cancer cells after down-regulation of eIF4E.4)After cell transfection(si-e IF4E),Western blot was used to determine the relative expression of eIF4E protein and epithelial mesenchymal transition(EMT)related proteins in breast cancer cells after down-regulation of eIF4E.5)CCK-8 cell proliferation assay was used to analyze the effect of down regulating eIF4E on the proliferation activity of breast cancer cells.6)Cell scratch test was used to analyze the effect of down regulating eIF4E on the migration ability of breast cancer cells.7)Flow cytometry was used to analyze the effect of down regulating eIF4E on apoptosis of breast cancer cells.8)Bioinformatics database-Targetscan looks for whether eIF4E gene is the downstream target gene of miRNA-429 and its binding site,which is verified by double-luciferasereporting gene experiment.9)Overexpression/knockdown of miRNA-429 in breast cancer cells after cell transfection(miRNA-429 mimics or miRNA-429 inhibitors).The relative expression of miRNA-429 after transfection was measured by RT-q PCR to determine the transfection efficiency.10)RT q PCR was used to detect the relative expression of eIF4E m RNA in breast cancer cells with different miRNA-429 expression levels.11)Western blot was used to detect the relative expression of eIF4E protein and epithelial mesenchymal transition(EMT)related proteins in breast cancer cells at different levels of miRNA-429 expression.12)CCK-8 cell proliferation assay was used to analyze the effect of overexpression/knockdown of miRNA-429 on the proliferation of breast cancer cells.13)Cell scratch test was used to analyze the effect of overexpression/knockdown of miRNA-429 on the migration ability of breast cancer cells.Results:1)The expression of eIF4E in breast cancer tissues and breast cancer cell lines increased compared with normal tissues and breast epithelial cells adjacent to breast cancer.2)After down-regulation of eIF4E,the proliferation,migration,anti apoptosis and EMT behavior of breast cancer cells were significantly inhibited.3)eIF4E was detected as a possible target gene downstream of miRNA-429 in Target Scan database.4)The double luciferase reporter gene verified that the "CAGUAUU" region in the target gene eIF4E m RNA 3 ’-UTR can bind with miRNA-429 and target the expression of eIF4E.5)Overexpression of miRNA-429 in breast cancer cells can significantly inhibit the expression of eIF4E m RNA and protein,as well as the process of EMT.6)Overexpression of miRNA-429 can significantly inhibit the proliferation and migration of breast cancer cells.Conclusion: miRNA-429 inhibits the proliferation,migration and other malignant behaviors of breast cancer cells by regulating the expression of target gene eIF4E.