MiR-124 Regulates EMT Based On ZEB2 Target To Inhibit Invasion And Metastasis In Triple-negative Breast Cancer

Objectives:Triple-negative breast cancer is the most common breast cancer subtype in young breast cancer patients and is characterized by a lack of progesterone receptor,estrogen receptor and human epidermal growth factor receptor 2 gene.It is highly invasive,and the clinical treatments are not satisfactory.Studying genes that regulate the malignant biological characteristics of triple-negative breast cancer will help reveal the pathogenesis of triple-negative breast cancer,and provide an ideal therapeutic target for gene therapy.miRNAs are small,non-coding RNAs of approximately 22-25 nucleotides in length that are capable of degrading or inhibiting translation of a target gene by binding to the 3’ UTR region of a target gene.miRNA can regulate the expression of a variety of tumor-associated genes in cells,and plays an important role in regulating cell cycle,cell proliferation,cell differentiation,apoptosis,inflammatory response and tumor invasion and metastasis.miR-124 is one of the earliest highly conserved miRNAs ever discovered.Many studies have pointed out that miR-124 is closely related to the occurrence and development of various tumors.However,the function and mechanism of miR-124 in triple-negative breast cancer are not clear.ZEB2 is a transcriptional regulator containing two zinc finger structural homology domains,which not only inhibits E-cadherin,but also inhibits other epithelial markers involved in cell polarity,cell tight junctions,gap junctions,and desmosome.ZEB2 can promote the invasion and metastasis of a variety of malignant tumor cells,which plays an important role in the process of tumor cell epithelial-mesenchymal transition.This study examined the expression of miR-124 in triple-negative breast cancer tissues and cells,and observed cell proliferation,colony formation,invasion,and EMT phenotype by overexpressing miR-124 in triple-negative breast cancer cells.Further study of the molecular mechanism of miR-124 involved in the regulation of triple-negative breast cancer cells provides experimental evidence for the study of new targets for the treatment of triple-negative breast cancer.Methods:1.Identification of miR-124 expression level in triple negative breast cancerThe mRNA expression levels of miR-124 were detected by quantitative real time PCR in triple negative breast cancer tissues and cells2.Fuctional analysis of miR-124 in triple negative breast cancer in vitromiR-124 was overexpressed in the triple negative breast cancer cell lines MDA-MB-231 and BT549,and the transfection efficiency was detected by real-time PCR.The effect of miR-124 on the proliferation of triple-negative breast cancer cells was detected by CCK8 assay.The effect of miR-124 on colony formation of triple-negative breast cancer cells was observed by plate cloning assay.The effect of miR-124 on the invasive ability of triple negative breast cancer cells was observed by Transwell invasion assay.3.The molecular mechanism of miR-124 on the invasive ability of triple negative breast cancer cellsBioinformatics software predicts that ZEB2 may be a direct target gene for miR-124,Overexpression of miR-124 inhibits ZEB2 by real-time PCR and Western blot;The dual luciferase reporter assay confirmed that miR-124 can be combined with the 3’ UTR region of ZEB2;The expressions of E-cadherin,N-cadherin and Vimentin mRNA and protein were texted by real-time PCR and Western blot.Transwell invasion assay analysis miR-124 inhibits the invasion of triple negative breast cancer cells by targeting ZEB2.Results:The expression of miR-124 was down-regulated in triple-negative breast cancer tissues and cells.Overexpression of miR-124 inhibited the proliferation,colony formation and invasion ability of triple negative breast cancer cells.Bioinformatics analysis indicated that miR-124 can bind to the 3’UTR region of ZEB2;Dual luciferase reporter assay further verified that miR-124 binds to the 3’UTR region of ZEB2;Overexpression of miR-124 can inhibit the expression of ZEB2;Overexpression of miR-124 can inhibit epithelialmesenchymal transition in triple-negative breast cancer cells;Overexpression of ZEB2 reversed the inhibitory effect of miR-124 on the invasive ability of triple negative breast cancer cells.Conclusions:miR-124 is down-regulated in triple-negative breast cancer tissues and cells;miR-124 inhibits proliferation,colony formation and invasion ability of triple negative breast cancer cells;miR-124 inhibits the invasion of triple negative breast cancer cells by targeting ZEB2 to affect epithelial-mesenchymal transition.