摘要
目的研究RhoA小干扰RNA对人结直肠癌细胞LoVo生物学特性的影响。方法构建针对RhoA mRNA的干扰质粒pGPU6/GFP/Neo-shRNA-RhoA并将其转染LoVo细胞。实时定量RT-PCR和Western印迹检测RhoA mRNA和蛋白的表达量;肿瘤细胞黏附实验检测细胞黏附能力的改变及Matrigel侵袭实验检测细胞侵袭力的改变。结果 LoVo细胞转染pshRNA-RhoA后,RhoA mRNA表达量明显下降,蛋白表达水平亦显著降低。细胞黏附实验和Matrigel侵袭实验结果显示,RhoA重组质粒转染组黏附率明显下降,细胞侵袭力明显下降。结论构建的靶向RhoA的pshRNA-RhoA真核表达载体可以有效、特异地阻断结直肠癌LoVo细胞中RhoA基因的表达,阻断RhoA基因的表达能显著降低其侵袭、黏附能力。
Objective To study the effects of RhoA siRNA on the malignant phenotypes of human colorectal cancer cell line LoVo.Methods The siRNA expression vector pGPU6/GFP/Neo-shRNA-RhoA targeting the mRNA of RhoA and vector pGPU6/GFP/Neo-NC(as a control) were constructed,and then transfected into LoVo cells.The expression of Survivin was detected by real-time fluorescent quantitative polymerase chain reaction and Western blot.The malignant phenotypes of transfected LoVo cells,including invasive activities and adhesive capabilities,were analyzed.Results RhoA mRNA and protein level were decreased after the pshRNA-RhoA transfection.The cell adhesion rates significantly decreased in the cells transfected with pshRNA-RhoA.The migrating number of LoVo cells((26.5±0.9)) transfected with pshRNA-RhoA was also significantly decreased as compared with the control group(53.7±1.4).Conclusions The sequence-specific shRNA against RhoA constructed in the study can blocking the expression of RhoA in LoVo cell effectively and specifically;Blocking the expression of RhoA in LoVo cells transfected with pshRNA-RhoA can reduce their invasive and adhesive capabilities.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2011年第15期2890-2892,共3页
Chinese Journal of Gerontology
基金
湖南省科学技术厅资助项目(2008FJ3160)