微小RNA-466靶向调控Twist1影响宫颈癌SiHa细胞增殖和迁移

MicroRNA-466 targeted regulation of Twist1 on the proliferation and migration of cervical cancer SiHa cells

目的研究微小RNA-466(miR-466)靶向调控碱性螺旋-环-螺旋转录因子1(Twist1)对宫颈癌SiHa细胞增殖和迁移的影响和机制。方法本研究起止时间为2019年1—10月。宫颈癌Caski、SiHa、C33A细胞购自上海沪震生物科技有限公司;正常宫颈上皮End1/E6E7细胞购自上海雅吉生物科技有限公司。定量即时聚合酶链锁反应(qRT-PCR)方法测定宫颈癌细胞中miR-466表达变化。在宫颈癌SiHa细胞中转染miR-466模拟物(miR-466 mimics),噻唑蓝(MTT)方法检测细胞增殖变化,Transwell小室检测细胞迁移和侵袭能力变化,蛋白质印迹法(Western blotting)检测细胞中波形蛋白(Vimentin)和上皮钙黏素(E-cadherin)蛋白表达变化。在线靶基因预测软件预测miR-466的靶基因可能为Twist1,荧光素酶报告系统鉴定靶向关系。将Twist1过表达载体和miR-466 mimics共转染到宫颈癌SiHa细胞中,检测细胞增殖、迁移和侵袭能力变化。结果正常宫颈上皮End1/E6E7细胞和宫颈癌Caski、SiHa、C33A细胞中miR-466表达水平依次为:(1.00±0.09)、(0.58±0.06)、(0.20±0.03)、(0.45±0.04);宫颈癌细胞系Caski、SiHa、C33A中miR-466表达水平低于End1/E6E7细胞,差异有统计学意义(P<0.0001)。与miR-NC组比较,miR-466组SiHa细胞细胞吸光度(0.29±0.04)比(0.45±0.02)降低,细胞侵袭数目[(81.46±6.35)个比(117.95±11.62)个]和迁移数目[(90.17±7.43)个比(147.15±12.04)个]下降,Vimentin蛋白表达水平降低,E-cadherin蛋白表达水平升高,差异有统计学意义(P<0.001)。与miR-466+pCDNA3.1组比较,miR-466+pCDNA3.1-Twist1组宫颈癌SiHa细胞吸光度(0.41±0.04)比(0.30±0.03)升高,细胞迁移数目[(127.34±10.26)个比(93.05±6.84)个]和侵袭数目[(108.94±10.24)个比(83.26±5.17)个]增加,Vimentin、Twist1蛋白表达增多,E-cadherin蛋白表达减少,差异有统计学意义(P<0.05)。上调miR-466靶向抑制Twist1表达。结论miR-466靶向抑制Twist1降低宫颈癌SiHa细胞增殖和迁移能力。

Objective To study the effect and mechanism of microRNA-466(miR-466)targeted regulation of Twist basic helix-loophelix transcription factor 1(Twist1)on the proliferation and migration of cervical cancer SiHa cells.Methods This study began in January 2019 and ended in October 2019.Cervical cancer Caski,SiHa,and C33A cells were purchased from Shanghai Huzhen Biotechnology Co.,Ltd.;normal cervical epithelial End1/E6E7 cells were purchased from Shanghai Yaji Biotechnology Co.,Ltd.Quantitative real time polymerase chain reaction(qRT PCR)was used to detect the expression of miR-466 in cervical cancer cells.miR-466 mimics was transfected into SiHa cells,methylthiazolyldiphenyl-tetrazolium bromide(MTT)method was used to detect cell proliferation,Transwell cells were used to detect cell migration and invasion,Western blotting was used to detect the expression of vimentin and epithelical cadherin(E-cadherin).Online target gene prediction software predicted that the target gene of miR-466 might be Twist1,the target relationship was identified by luciferase report system.Twist1 overexpression vector and miR-466 mimics were co transfected into cervical cancer SiHa cells to detect cell proliferation,migration and invasion.Results The expression levels of miR-466 in normal cervical epithelial End1/E6E7 cells and cervical cancer Caski,SiHa,and C33A cells were:(1.00±0.09),(0.58±0.06),(0.20±0.03),(0.45±0.04);the expression level of miR-466 in the cancer cell lines Caski,SiHa,and C33A was lower than that in End1/E6E7 cells,and the difference was statistically significant(P<0.0001).Compared with the miR-NC group,the cell A value of SiHa cells in the miR-466 group(0.29±0.04 vs.0.45±0.02)was decreased,the number of cell invasion[(81.46±6.35)vs.(117.95±11.62)]and the number of migration[(90.17±7.43)vs.(147.15±12.04)]were decreased,Vimentin protein expression level was decreased,E-cadherin protein expression level was increased,the difference was statistically significant(P<0.001).Compared with the miR-466+pCDNA3.1 group,the A value of cervical cancer SiHa cells in the miR-466+pCDNA3.1-Twist1 group(0.41±0.04 vs.0.30±0.03)was increased,and the number of cell migration[(127.34±10.26)vs.(93.05±6.84)and the number of invasion[(108.94±10.24)vs.(83.26±5.17)]were increased,Vimentin and Twist1 protein expression were increased,E-cadherin protein expression was decreased,the difference was statistically significant(P<0.05).Up-regulation of miR-466 targeted inhibition of Twist1 expression.Conclusion miR-466 targeted inhibition of Twist1 reduced the proliferation and migration of SiHa cells.