The Role And Mechanism Of MiR-320 In Breast Cancer

BackgroudBreast cancer is the most common malignancy in women.Because of the progress in early screening and treatment strategy,the incidence and mortality rate of breast cancer have been decreased in the past few years,but it is still a major threat to women's physical and mental health.Research on the development of breast cancer related genes can help to identify new prognostic markers and therapeutic targets.In recent years,much more studies have been demonstrated that alternation of miRNAs expression play an important role in the development,progression and prognosis of breast cancer.Therefore,miRNA is a potential marker for early diagnosis and treatment in patients with breast cancer,and is more likely to be a potential target for molecular targeted therapy of breast cancer.ObjectiveTo investigate the relationship between miR-320 and breast cancer development,and evaluate the clinical value of miR-320 on the diagnosis and prognostic prediction in patients with breast cancer.To identify the role and mechanism of miR-320 in breast cancer development and progression.Methods1.RT-qPCR analysis was used to detected the miR-320 expression in 180 cases of primary breast cancer tissues and 15 cases of paired normal breast tissues.The paired t test was used to compare the differences of miR-320 levels between the breast cancer and normal groups.Chi-square test was used to compare the differences between miR-320 expression and clinicopathological factors.Kaplan-Meier survival curve was used to analyze the relationship between miR-320 expression and overall survival(OS)or disease-free survival(DFS),Cox proportional hazard regression Model to evaluate its prognostic value.2.RT-qPCR was used to detect the expression of miR-320 in various breast cancer cell lines with different metastatic abilities.3.miR-320 mimcs and miR-320 inhibitor were transfected to MDA-MB-231 and MCF7,respectively.MTT and colony formation assays were used to evaluate the effect of miR-320 on proliferation in breast cancer cell lines.Transwell assay was used to evaluate the effect of miR-320 on the invasion in breast cancer cell lines.Flow cytometry was used to determine effect of miR-320 on the cell cycle distribution and apoptosis.4.The regulatory activity of miR-320 on down-stream target gene SOX4 was analyzed by double-luciferase reporter system and site-directed mutagenesis.RT-qPCR and Western blot were used to detect the expression of SOX4 in miR-320-expressed or-depleted breast cancer cell lines.5.The expression of epithelial-mesenchymal transition(EMT)markers E-cadherin,and Vimentin were detected by RT-qPCR and Western blot in miR-320-expressed or-depleted breast cancer cell lines.Results1.The expression of miR-320 is down-regulated in breast cancer tissues compared to the matched normal tissues(P <0.001).The patients with low miR-320 expression have an advanced stage(P = 0.043)and a poor outcome(P = 0.025).Furthermore,the expression of miR-320 is an independent prognostic predictor for 3-year DFS in patients with breast cancer.2.miR-320 is highly expressed in MCF7 and T47 D with lower invasive and mobility ability,whereas miR-320 is lower expressed in MDA-MB-231 and MDA-MB-468 with higher invasive and mobility ability.3.Overexpression of miR-320 inhibits cell proliferation and invasion,and promotes cell apoptosis in breast cancer cell line MDA-MB-231,whereas depletion of miR-320 promotes cell proliferation and invasion in breast cancer cell line MCF7.The flow cytometry showed that overexpression of miR-320 leads to cell cycle arrest at G1 phase in MDA-MB-231,whereas miR-320-depleted MCF7 cells have a higher percentage of cells at S phase.4.Bioinformatic analysis predicts that there is two miR-320 binding sites on the SOX4 3'-UTR region.The dual-luciferase reporter system demonstrates that miR-320 can reduce the wide-type SOX4 3'-UTR activity,whereas miR-320 cannot reduce the mutated SOX4 3'-UTR activity.Overexpression of miR-320 down-regulates the expression of SOX4 at both m RNA and protein levels,whereas depletion of SOX4 up-regulates the expression of SOX4 at both m RNA and protein levels.5.The expression of epithelial marker E-cadherin is increased,whereas the expression of mesenchymal marker Vimentin is decreased in miR-320-overexpressed MDA-MB-231 cells.In contrast,the expression of epithelial marker E-cadherin is decreased,whereas the expression of mesenchymal marker Vimentin is increased in miR-320-depleted MCF7 cells.6.The expression of miR-320 is down-regulated in MCF7 cells after treatment with TGF?1.Furthmore,overexpression of miR-320 inhibits cell invasion in TGF?1-treated MCF7 cells.Conclusion1.The expression of miR-320 is an independent prognostic factor for patients with breast cancer.2.miR-320 inhibits the proliferation and invasion of breast cancer cells,and promotes cell apoptosis by down-regulation of SOX4 expression.3.miR-320 leads to cell cycle arrest at G1 phase.4.miR-320 inhibits the EMT phenotype in breast cancer cells.