Wang P, Liu S, Cheng B, Wu X Z, Ding S S, Xu L, Liu Y, Duan L, Sun S Z
Department of Pathology, the General Hospital of PLA Rocket Force, Beijing 100088, China.
Zhonghua Bing Li Xue Za Zhi. 2017 Mar 8;46(3):187-192. doi: 10.3760/cma.j.issn.0529-5807.2017.03.009.
To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.
研究细胞周期蛋白D1过表达对宫颈鳞状细胞癌SiHa细胞增殖和分化的影响，并探讨相关信号分子。设计引物扩增细胞周期蛋白D1基因全长，通过PCR扩增细胞周期蛋白D1基因以构建pcDNA3.1质粒载体。然后将构建体转染到SiHa细胞中，建立细胞周期蛋白D1稳定过表达的细胞系，分别通过RT-PCR和蛋白质印迹法检测细胞周期蛋白D1基因和蛋白表达。采用MTT法绘制细胞生长曲线。通过RT-PCR和蛋白质印迹法检测转染细胞中CK7、E-钙黏蛋白、波形蛋白、Snail基因和蛋白表达。采用RT-PCR检测细胞周期蛋白依赖性激酶4（CDK4）、细胞周期蛋白依赖性激酶2（CDK2）、p21、p27、细胞周期蛋白E、视网膜母细胞瘤蛋白（Rb）、E2F、E6/E7和Ki-67等增殖和分化相关基因的mRNA表达。细胞同步化后，采用RT-PCR检测细胞周期不同时间点细胞周期蛋白D1和p21的mRNA表达。成功建立了细胞周期蛋白D1过表达的G-3细胞系。G-3细胞的生长曲线和Ki-67 mRNA表达加快。波形蛋白和Snail在基因和蛋白水平的表达均显著增加，而E-钙黏蛋白、CK7基因和蛋白表达显著降低，表明G-3细胞发生了上皮-间质转化。同时，细胞周期蛋白D1、CDK4、CDK2、p21、p27、细胞周期蛋白E、E2F和Rb的mRNA表达增加，而E6/E7和p16无明显变化。p21和细胞周期蛋白D1的表达趋势在细胞周期不同时间点波动时几乎相同。基因转染诱导的细胞周期蛋白D1过表达促进SiHa细胞增殖和上皮-间质转化。该过程伴随着CDK4、CDK2、p21、p27和细胞周期蛋白E基因的上调。p21表达与细胞周期蛋白D1同步增加，提示其通过影响G-3细胞中波形蛋白的表达在上皮-间质转化中发挥调节作用。
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