miR-301a靶向雌激素受体信号通路对乳腺癌细胞侵袭及上皮-间质转化的影响

Effects of miR-301a targeting estrogen receptor signaling pathway on the invasion of breast cancer cells and epithelial-mesenchymal transition

目的通过检测乳腺癌细胞中微小RNA-301a(miR-301a)、雌激素受体α(ERα)表达情况,探讨miR-301a调控乳腺癌细胞侵袭作用机制及对上皮-间质转化(EMT)的影响。方法体外培养人乳腺癌细系MCF7,将细胞分为control组(不做任何处理,正常培养)、miR-301a mimics组[转染miR-301a激动剂(miR-301a mimics)]、miR-301a inhibitiors组[转染miR-301a抑制剂(miR-301a inhibitiors)]、miR-301a MNC组[转染miR-301a激动剂阴性对照试剂(miR-301a MNC)]、miR-301a INC组[转染miR-301a抑制剂阴性对照试剂(miR-301a INC)]。用Transwell小室检测细胞侵袭情况;双荧光素酶报告实验验证miR-301a与ERα的靶向关系;荧光实时定量PCR(qRT-PCR)法检测各组miR-301a mRNA、ERαmRNA表达水平;免疫印迹法(Western blot)检测各组细胞ERα、孕酮受体(PR)、乳腺癌雌激素调控蛋白(GREB1)及EMT标志蛋白N-粘钙蛋白(N-cadherin)、波形蛋白(Vimentin)表达水平。结果与control组相比,miR-301a mimics组侵袭细胞数、miR-301a、N-cadherin和Vimentin蛋白表达均升高(P<0.05),ERαmiRNA及蛋白、PR、GREB1表达均降低(P<0.05);miR-301a inhibitiors组侵袭细胞数、miR-301a、N-cadherin和Vimentin蛋白表达均降低(P<0.05),ERαmiRNA及蛋白、PR、GREB1表达水平均升高(P<0.05)。双荧光素酶报告基因检测显示,与miR-301a NC+ERα-3’UTR-WT组比较,miR-301a mimics+ERα-3’UTR-WT组荧光素酶活性降低(P<0.05)。结论miR-301a过表达可抑制ERα及相关通路蛋白表达,促进癌细胞侵袭和EMT,抑制miR-301a表达,可上调ERα及相关通路表达,抑制癌细胞侵袭和EMT。

Objective To investigate the expressions of miRNA-301a(miR-301a)and estrogen receptorα(ERα),and to exlore the effects of miR-301a targeting estrogen receptor signaling pathway on the invasion of breast cancer cells and epithelial-mesenchymal transition(EMT).Methods Human breast cancer cell line MCF7 cells were cultured in vitro and divided into control group(without any treatment),miR-301a mimics group[transfection miR-301a agonist(miR-301a mimics)],miR-301a inhibitiors group[transfection miR-301a inhibitor(miR-301a)inhibitiors)],miR-301a MNC group[transfection miR-301a agonist negative control reagent(miR-301a MNC)],miR-301a INC group[transfection miR-301a inhibitor negative control reagent(miR-301a INC)].Transwell cell was used to detect the invasion of cells;double luciferase report assay was used to verify the targeting correlation between miR-301a and ERα,and the expression levels of miR-301a and ERαmRNA were detected by quantitative real-time polymerase chain reaction(qRT-PCR);the expression levels of pathway-related proteins ERα,progesterone receptor(PR),gene regulated by estrogen in breast cancer protein(GREB1),EMT marker protein N-cadherin and Vimentin and other protein were detected by Western Blot(WB).Results Compared with those in control group,the number of invasive cells,and the expression levels of miR-301a,the N-cadherin and Vimentin protein in miR-301a mimics group were significantly increased(P<0.05),however,the expression levels of ERαmiRNA and pathway-related proteins ERα,PR,GREB1 were significantly decreased(P<0.05).And the numbers of invasive cells,and the expression levels of miR-301a,and the expression level of EMT markers N-cadherin and Vimentin in miR-301a inhibitors group were significantly decreased(P<0.05),however,the expression levels of ERαmiRNA and pathway-related proteins ERα,PR,GREB1 were significantly increased(P<0.05).Double luciferase reporter gene detection showed that the activity of luciferase in miR-301a mimics+ERα-3'UTR-WT group was significantly lower than that in miR-301a NC+ERα-3'UTR-WT group(P<0.05).Conclusion The overexpression of miR-301a can inhibit the expression of ERαand related pathway proteins,promote the invasion of cancer cells and EMT,inhibit the expression of miR-301a,and up regulate the expression of ERαand related pathway,so as to inhibit the invasion of cancer cells and EMT.