miR-429对乳腺癌细胞增殖和迁移的影响及其机制

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

目的探讨miR-429对乳腺癌细胞增殖和迁移的影响及其机制。方法采用实时荧光定量PCR(RT-qPCR)法和免疫印迹法,分别检测乳腺癌MCF-7、MDA-MB-231、MDA-MB-468、BT-549细胞及正常乳腺上皮细胞MCF-10A的eIF4E mRNA和蛋白相对表达量。在MCF-7细胞中转染miR-429 mimics(A组)、mimics NC(B组)、miR-429 inhibitors(C组)及inhibitors NC(D组),采用RT-qPCR法、CCK8法、细胞划痕实验和免疫印迹法检测A~D组细胞miR-429表达情况、细胞增殖和迁移情况、eIF4E mRNA和蛋白相对表达量。在293T细胞中转染eIF4E-3′UTR-Wt+miR-429 mimics(E组)、eIF4E-3′UTR-Wt+mimics NC(F组)、eIF4E-3′UTR-Mut+miR-429 mimics(G组)、eIF4E-3′UTR-Mut+mimics NC(H组),检测E~H组细胞相对荧光素酶活性,分析miR-429对eIF4E的靶向调控作用。结果RT-qPCR与免疫印迹检测结果显示,MCF-7、MDA-MB-231、MDA-MB-468、BT-549细胞中的eIF4E mRNA以及蛋白的相对表达量均明显高于正常乳腺上皮细胞MCF-10A(F=74.414、1981.243,P<0.01)。RT-qPCR法检测结果显示,A组细胞的miR-429相对表达量明显高于B组(t=25.390,P<0.01),而eIF4E mRNA相对表达量明显低于B组(t=-6.363,P<0.05),C组细胞miR-429相对表达量明显低于D组(t=-4.652,P<0.05),而eIF4E mRNA相对表达量明显高于B组(t=-2.928,P<0.05)。CCK8实验检测结果显示,与B组细胞相比,A组细胞第24、48、72小时时的增殖活力明显降低(F=26.148~40.997,P<0.01);与D组细胞相比,C组细胞第24、48、72小时增殖活力明显增高(F=6.410~82.593,P<0.05)。细胞划痕实验显示,A组细胞愈合率明显低于B组(t=-22.584,P<0.01),C组细胞愈合率明显高于D组(t=11.464,P<0.01)。免疫印迹法检测结果显示,A组细胞中eIF4E蛋白相对表达量明显低于B组(t=-20.355,P<0.01),C组细胞eIF4E蛋白相对表达量明显高于D组(t=3.622,P<0.01)。双荧光素酶报告基因实验结果显示,各组细胞相对荧光素酶活性比较差异具有显著性(F=366.823,P<0.05),而且E组细胞相对荧光素酶活性显著低于F组(t=-42.961,P<0.01)。结论miR-429或通过靶向调控eIF4E基因进而抑制乳腺癌细胞的增殖和迁移。

Objective To investigate the effect of miR-429 on the proliferation and migration of breast cancer cells and its mechanism.Methods RT-qPCR and Western blotting were used to measure the relative mRNA and protein expression levels of eIF4E in breast cancer MCF-7,MDA-MB-231,MDA-MB-468,and BT-549 cells and normal breast epithelial MCF-10A cells.MCF-7 cells were transfected with miR-429 mimics(group A),mimics NC(group B),miR-429 inhibitors(group C),and inhibitors NC(group D),and RT-qPCR,CCK-8 assay,wound healing assay,and Western blotting were used to measure the expression of miR-429,cell proliferation and migration,and the relative mRNA and protein expression levels of eIF4E in groups A-D.The 293T cells were transfected with eIF4E-3′UTR-Wt+miR-429 mimics(group E),eIF4E-3′UTR-Wt+mimics NC(group F),eIF4E-3′UTR-Mut+miR-429 mimics(group G),and eIF4E-3′UTR-Mut+mimics NC(group H),and relative luciferase activity was measured for groups E-H.The targeted regulatory effect of miR-429 on eIF4E was analyzed.Results RT-qPCR and Western blotting showed that the relative mRNA and protein expression levels of eIF4E in MCF-7,MDA-MB-231,MDA-MB-468,and BT-549 cells were significantly higher than those in normal breast epithelial MCF-10A cells(F=74.414,1981.243,P<0.01).RT-qPCR showed that compared with group B,group A had a significantly higher relative expression level of miR-429(t=25.390,P<0.01)and a significantly lower relative mRNA expression level of eIF4E(t=-6.363,P<0.05);group C had a significantly lower relative expression level of miR-429 than group D(t=-4.652,P<0.05)and a significantly higher relative mRNA expression level of eIF4E than group B(t=-2.928,P<0.05).CCK8 assay showed that compared with group B,group A had a significant reduction in proliferation activity at 24,48,and 72 hours(F=26.148-40.997,P<0.01),and compared with group D,group C had a significant increase in proliferation activity at 24,48,and 72 hours(F=6.410-82.593,P<0.05).Wound healing assay showed that group A had a significantly lower healing rate than group B(t=-22.584,P<0.01),and group C had a significantly higher healing rate than group D(t=11.464,P<0.01).Western blotting showed that group A had a significantly lower relative protein expression level of eIF4E than group B(t=-20.355,P<0.01),and group C had a significantly higher relative protein expression level of eIF4E than group D(t=3.622,P<0.01).Dual-luciferase reporter assay showed that there was a significant difference in relative luciferase activity between groups(F=366.823,P<0.05),and group E had a significantly lower relative luciferase activity than group F(t=-42.961,P<0.01).Conclusion This study shows that miR-429 may inhibit the proliferation and migration of breast cancer cells through targeted regulation of the eIF4E gene.