miR-429对乳腺癌细胞增殖的影响及其作用机制

EFFECT OF miR-429 ON THE PROLIFERATION OF BREAST CANCER CELLS AND ITS MECHANISM

目的探讨miR-429对乳腺癌细胞增殖的影响及其具体作用机制。方法体外培养人乳腺癌细胞MDA-MB-231,根据转染成分不同将细胞分为转染mimics NC组(A组)、空白对照组(B组)、转染inhibitors组(C组)、转染inhibitors NC组(D组)和转染miR-429 mimics组(E组),应用MTT法检测miR-429表达对人乳腺癌细胞MDA-MB-231增殖活力的影响,通过Western blot方法检测miR-429对低密度脂蛋白受体相关蛋白1(LRP1)表达的影响;采用microRNA靶基因预测软件TargetScan 7.1预测miR-429与LRP1潜在的互补结合位点;以pSI-Check2为工具载体构建h-LRP1-3UTR-wt及h-LRP1-3UTR-mu双荧光素酶报告基因质粒,根据转染成分不同将293T细胞分为转染h-LRP1-3UTR-mu与NC mimics组(F组)、转染h-LRP1-3UTR-mu与miR-429 mimics组(G组)、转染h-LRP1-3UTR-wt与NC mimics组(H组)、转染h-LRP1-3UTR-wt与miR-429 mimics组(I组),检测每组293T细胞中荧光素酶的活性,分析miR-429对LRP1的靶向调控作用。结果MTT实验结果示,在细胞培养的第2~4天,E组细胞的增殖活力明显低于B、C、D组(F=4.53~19.12,P<0.05);Western blot检测结果示,E组细胞LRP1蛋白表达量明显低于A、B、C、D组(F=6.26,P<0.05);TargetScan 7.1软件预测结果显示人miR-429与LRP1基因3′非翻译区(3′UTR)存在互补结合位点;荧光素酶活性检测结果显示,与H组相比I组细胞荧光素酶活性下调(t=39.78,P<0.01)。结论miR-429通过对LRP1的靶向调控作用来抑制乳腺癌细胞的增殖,其对LRP1的靶向调控机制是通过与LRP1基因3′UTR互补结合实现的。

Objective To investigate the effect of miR-429 on the proliferation of breast cancer cells and its mechanism of action.Methods Human breast cancer cells MDA-MB-231 were cultured in vitro and then divided into group A(transfected with mimics NC),group B(blank control group),group C(transfected with inhibitors),group D(transfected with inhibitors NC),and group E(transfected with miR-429 mimics)according to the transfection component.MTT assay was used to observe the effect of miR-429 expression on the proliferation activity of human breast cancer MDA-MB-231 cells;Western blot was used to observe the effect of miR-429 on the expression of low-density lipoprotein receptor-related protein 1(LRP1);the microRNA target gene prediction software TargetScan 7.1 was used to predict the potential complementary binding sites between miR-429 and LRP1.The h-LRP1-3UTR-wt and h-LRP1-3UTR-mu dual-luciferase reporter plasmids were constructed with pSI-Check2 as the vector,and according to the transfection component,293T cells were divided into group F(transfected with h-LRP1-3UTR-mu and NC mimics),group G(transfected with h-LRP1-3UTR-mu and miR-429 mimics),group H(transfected with h-LRP1-3UTR-wt and NC mimics),and group I(transfected with h-LRP1-3UTR-wt and miR-429 mimics);luciferase activity was measured for each group to analyze the targeted regulatory effect of miR-429 on LRP1.Results MTT assay showed that on days 2-4 of cell culture,group E had significantly lower cell proliferation activity than groups B,C,and D(F=4.53-19.12,P<0.05);Western blot showed that group E had significantly lower protein expression of LRP1 than groups A,B,C,and D(F=6.26,P<0.05);TargetScan 7.1 software predicted that there was a complementary binding site between human miR-429 and 3′untranslated region(3′UTR)of the LRP1 gene;the results of luciferase activity showed that compared with group H,group I had significant downregulation of luciferase activity(t=39.78,P<0.01).Conclusion miR-429 can inhibit the proliferation of breast cancer cells by targeted regulation of LRP1,which is realized through complementary binding between miR-429 and 3′UTR of the LRP1 gene.