miR-1246对人宫颈癌上皮-间质转化及其增殖、侵袭和迁移能力影响的初步研究

Preliminary study on the effect of miR-1246 on epithelial-mesenchymal transition,proliferation,invasion and migration of cervical cancer

目的探讨miR-1246水平变化对人宫颈癌SiHa细胞上皮-间质转化(EMT)及其增殖、侵袭和迁移能力的影响。方法体外向SiHa细胞分别转染miR-1246模拟物(miR-1246-mimics组)及阴性对照(mimics-NC组)、miR-1246抑制物(miR-1246-inhibitor组)及阴性对照(Inhibitor-NC组),以未作处理的SiHa细胞为空白对照组。通过流式细胞术和实时荧光定量PCR(qRT-PCR)检测转染效率。采用Western blotting检测转染后细胞E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)及Twist-2蛋白的表达水平。通过CCK-8法、流式细胞术、Transwell小室实验和划痕实验检测转染后SiHa细胞的增殖、凋亡、侵袭和迁移能力。结果流式细胞术结果显示,各组细胞转染效率均在90%以上。qRT-PCR结果显示,与空白对照组和阴性对照组比较,miR-1246-mimics组miR-1246的相对表达量显著增高,而miR-1246-inhibitor组明显降低(P<0.05)。Western blotting结果显示,与阴性对照组比较,miR-1246-mimics组E-cadherin蛋白表达水平降低而N-cadherin、Vimentin和Twist-2蛋白表达水平显著增加(P<0.05),而miR-1246-inhibitor组E-cadherin蛋白表达水平增加而N-cadherin、Vimentin和Twist-2蛋白表达水平显著降低(P<0.05)。与阴性对照组比较,miR-1246-mimics组细胞增殖、侵袭和迁移能力均显著提高(P<0.05),而细胞凋亡率显著降低(P<0.05);与阴性对照组比较,miR-1246-inhibitor组细胞增殖、侵袭和迁移能力均显著降低(P<0.05),而细胞凋亡率显著提高(P<0.05)。结论过表达miR-1246可能通过EMT促进宫颈癌SiHa细胞的增殖、侵袭及迁移并诱导细胞凋亡。

Objective To investigate the effect of miR-1246 on epithelial-mesenchymal transition(EMT)and its proliferation,invasion and migration of human cervical cancer SiHa cells.Methods In vitro,SiHa cells were transfected with miR-1246 simulant(miR-1246-mimics group)and negative control(mimics-NC group),miR-1246 inhibitor(miR-1246-inhibitor group)and negative control(Inhibitor-NC group),and untreated SiHa cells were used as the blank control group.The transfection efficiency was detected by flow cytometry and real-time quantitative PCR(qRT-PCR).Western blotting was used to detect the expression levels of E-cadherin,N-cadherin,Vimentin and Twist-2 in SiHa cells after transfection.CCK-8,flow cytometry apoptosis experiment,Transwell chamber and scratch experiment were used to compare the proliferation,apoptosis,invasion and migration abilities of SiHa cells after transfection.Results Flow cytometry results showed that the transfection efficiency of cells in each group was above 90%.The qRT-PCR results indicated that compared with the blank control and the negative control group,the relative expression of miR-1246 in the miR-1246-mimics group was significantly higher,while the miR-1246-inhibitor group was significantly lower(P<0.05).Western blotting showed that compared with the negative control group,the expression levels of E-cadherin protein decreased and the expression levels of N-cadherin,Vimentin and Twist-2 protein increased significantly in miR-1246-mimics group(P<0.05),while the expression levels of E-cadherin protein increased and the expression levels of N-cadherin,Vimentin and Twist-2 protein decreased significantly in miR-1246-inhibitor group(P<0.05).Compared with the negative control group,the ability of proliferation,invasion and migration of cells in miR-1246-mimics group was significantly increased(P<0.05),while the apoptosis rates were significantly reduced(P<0.05).Compared with the negative control group,the ability of proliferation,invasion and migration of miR-1246-inhibitor group was significantly decreased(P<0.05),while the apoptosis rates were significantly increased(P<0.05).Conclusion Overexpression of miR-1246 promotes the proliferation,invasion,migration and induces cell apoptosis of cervical cancer SiHa cells through EMT.