MicrornA-497 Targets SMAD 7 And Has A Tumour Suppressive Effect In Breast Cancer

ObjectiveBreast cancer is one of the most serious problems of oncology also it is the leading cause of death of women in many countries without geographic restrictions.Every year nearly 1.2 million women suffer from breast cancer worldwide, and500,000 people died of breast cancer. In our country, breast cancer incidence is growing at an annual rate of 3% recent years, becoming the leading cause of death of women in city. Latest studies have shown that SMAD 7 played a major role in breast cancer during tumour development and progression. In the current study, we aimed to examine the relationship between microRNA-497(miR-497) and SMAD 7 and its function in MDA-MB-231 breast cancer cells and MCF-7 breast cancer cells. Based on miR target analysis using the websites targetscan, miRanda, miRGen and miRBase,SMAD 7 was a candidate target of miR-497. To test whether miR-497 could bind to3’-untranslated region(3’-UTR) of SMAD 7 mRNA, luciferase reporter assay was conducted. Quantitative polymerase chain reaction and Western blot assays were used to confirm the dual-luciferase results. MTT assays, colony formation assays and cell cycle assays were utilized to examine examine the potential function of miR-497 in MDA-MB-231 breast cancer cells and MCF-7 breast cancer cells.MethodsBased on miR target analysis using the websites targetscan, miRanda, miRGen and miRBase, SMAD 7 was a candidate target of miR-497. To test whether miR-497 could bind to 3’-untranslated region(3’-UTR) of SMAD 7 mRNA, luciferase reporter assay was conducted. Quantitative polymerase chain reaction and Western blot assays were used to confirm the dual-luciferase results. MTT assays, colony formation assays and cell cycle assays were utilized to examine examine the potential function of miR-497 in MDA-MB-231 breast cancer cells and MCF-7 breast cancer cells.Results1、MiR-497 directly targets SMAD 7 and downregulates its expression in MDA-MB-231 cells and MCF-7 cells. The predicted binding of miR-497 with SMAD7 3′-UTR was shown that SMAD 7 might be a potential target of miR-497.2、To detect whether mi R-497 directly targets SMAD 7, we used dual-luciferase reporter assays, as a consequence, the Renilla luciferase(RL) activity /firefly luciferase(FL) activity indicates the relative activity levels.3、 The results showed that SMAD 7 mRNA levels and SMAD 7 proteins were lower in mi R-497 overexpressing group compared with the negative control group,indicated that MiR-497 targets SMAD 7 and downregulates its expression in MDA-MB-231 cells and MCF-7 cells.4、Overexpression of miR-497 in MDA-MB-231 cells and MCF-7 cells inhibited cell proliferation. Compared with the negative control group, MDA-MB-231 cells and MCF-7 cells transfected with miR-497 showed significantly repression.5、Colony formation assays indicated that more colony formation in the group transfected with negative control compared with the 100 nM miR-497 group. All the results showed that overexpression of miR-497 in MDA-MB-231 cells and MCF-7cells inhibited cell proliferation.6、Overexpression of miR-497 in MDA-MB-231 cells and MCF-7 cells affected Cell cycle.The results demonstrated that overexpression of miR-497 in MDA-MB-231 cells and MCF-7 cells affected Cell cycle progression.ConclusionsThe results showed that MiR-497 was downregulated in breast cancer tissues compared with normol tissues. Dual-luciferase reporter assays manifestations that SMAD 7 is the target of miR-497. Quantitative polymerase chain reaction and Western blot assays validated that overexpression of miR-497 can reduce SMAD 7protein levels, miR-497 can inhibit cellular growth and cause a G1 arrest. In summary,MiR-497 act as a tumor suppressor gene in breast cancer. Overexpression of miR-497 inhibited cell proliferation and G1 cell cycle arrest were observed. This is potentially by targeting SMAD 7. The above results indicated that miR-497 could be considered an ideal therapeutic target for the disease development in breast cancer.