Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells
10.3760/cma.j.issn.0529-5807.2017.03.009
- VernacularTitle: cyclin D1过表达对子宫颈鳞状细胞癌SiHa细胞增殖及上皮间质转化的影响
- Author:
Ping WANG
1
;
Shan LIU
;
Bo CHENG
;
Xizhao WU
;
Shanshan DING
;
Lin XU
;
Ye LIU
;
Lian DUAN
;
Suozhu SUN
Author Information
1. Department of Pathology, the General Hospital of PLA Rocket Force, Beijing 100088, China
- Publication Type:Journal Article
- Keywords:
Cyclin D;
p21-activated kinases;
Cell proliferation;
Cell differentiation;
Reverse transcriptase polymerase chain reaction
- From:
Chinese Journal of Pathology
2017;46(3):187-192
- CountryChina
- Language:Chinese
-
Abstract:
Objective: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.
Methods: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.
Results: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.
Conclusions: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.
