RNA干扰沉默Ras同源类似物C对恶性黑色素瘤细胞侵袭转移的影响  

Impacts of RhoC silencing on invasion and metastasis of human melanoma cells

作　　者：王琪影[1] 王晓利[1] 刘林嶓[1] 

机构地区：[1]郑州大学第一附属医院整形外科,450052

出　　处：《中华实验外科杂志》2013年第9期1949-1951,共3页Chinese Journal of Experimental Surgery

基　　金：郑州市科技局科技攻关计划资助项目（10TGS484-12）

摘　　要：目的 观察靶向Ras同源类似物C（RhoC）的短发卡RNA（shRNA）质粒载体构建及RhoC基因沉默对人恶性黑色素瘤A375细胞侵袭、转移能力的影响.方法 构建3条质粒载体pGPU6/Neo-RhoC shRNA1、shRNA2和shRNA3,利用脂质体LlpofectamlneTM 2000转染A375细胞,Western blot检测转染后RhoC蛋白表达,并将最佳靶向序列质粒转染A375细胞,应用细胞黏附实验和Transwell小室细胞侵袭实验分别检测细胞黏附能力及侵袭能力.结果 构建的3条质粒载体pGPU6/Neo-RhoC shRNA1、shRNA2和shRNA3转染A375细胞后,RhoC蛋白表达水平分别为0.284 ±0.041、0.136±0.022和0.213 ±0.032,pGPU6/Neo-RhoC shRNA2为最佳靶向序列质粒；将pGPU6/Neo-RhoC shRNA2转染A375细胞,细胞对层粘连蛋白和纤维粘连蛋白的黏附能力、细胞穿膜细胞分别为0.473±0.085、0.324±0.023和50.000±14.526,较空白对照组和阴性对照组明显下降,差异有统计学意义（P＜0.05）.结论 利用RNAi技术成功构建pGPU6/Neo-RhoC shRNA质粒载体可显著下调体外培养的A375细胞RhoC蛋白的表达,且RhoC基因沉默后细胞的侵袭转移能力明显受到抑制.Objective To investigate the construction of RNA interterence vector targeting Ras homology C （RhoC）,as well as the effects of RhoC silencing on the invasion and metastasis of human melanoma cells A375.Methods Three pairs of pGPU6/Neo-RhoC short hairpin RNA （shRNA） 1,shRNA2 and shRNA3 vectors were constructed and transfected into A375 cells through Lipofectamine.The protein expression of RhoC was detected by using Western blotting,and the most effective sequence was selected based on the silencing effects.After A375 cells were transfected by using the most effective sequence,the adhesion to laminin and fibronectin was examined by using cell adhesion experiment,and the invasive capacity was tested by using Transwell chamber assay.Results pGPU6/Neo-RhoC shRNA1,shRNA2 and shRNA3 were constructed successfully and their sequences were proved to be accurate.After transfection into A375 cells,the protein expression of RhoC was 0.284 ±0.041,0.136 ±0.022 and 0.213 ±0.032 respectively.Among three pairs of pGPU6/Neo-RhoC shRNA vector,pGPU6/Neo-RhoC shRNA2 was the most effective sequence.After A375 cells were transfected with pGPU6/Neo-RhoC shRNA2,the adhesion to laminin and fibronectin,and the penetrating cells in the transfected group were 0.473 ± 0.085,0.324 ±0.023 and 50.000 ± 14.526,which were reduced as compared with those in the blank control group and negative control group （P ＜ 0.05）.Conclusion RhoC was silenced in A375 cells transfected with pGPU6/Neo-RhoC shRNA by using RNA interference technique,which inhibited the invasion and metastasis of A375 cells.

关 键 词：人黑色素瘤细胞 Ras同源类似物C RNA干扰 侵袭 转移 

分 类 号：R739.5[医药卫生—肿瘤]