RhoC沉默抑制卵巢癌细胞的生长及转移

RhoC miRNA inhibits the movement and invasiveness of ovarian cancer SKOV3 cells

目的:探讨RNA干扰技术沉默Ras homolgyC(RhoC)对卵巢癌细胞SKOV3细胞运动和侵袭力的影响。方法:利用Invitrogenis RNAi Designer软件设计并构建RhoC的干扰RNA(miRNA)载体,利用质脂体介导法将RhoC miRNA及非特异miRNA转染卵巢癌SKOV3细胞,经blasticdin进行稳定筛选出抗性克隆;将实验分为RhoC miRNA组(转染特异miRNA)、非特异miRNA组(转染非特异性miRNA)、空白对照组(不转染RhoC miRNA)。Western blot检测3组卵巢癌细胞中RhoC蛋白表达水平;同时检测对卵巢癌细胞体外运动及侵袭的抑制作用。结果:SKOV3细胞稳定转染后,转染特异性miRNA组RhoC蛋白表达量[0.1989(4257/21498)]与转染非特异miRNA组[2.142(38245/17855)]和空白对照组[2.604(48872/18768)]相比,差异均有统计学意义(P<0.01),而非特异性组与空白对照组之间差异无统计学意义(P>0.05);体外细胞运动实验表明,转染特异性miRNA组与转染非特异性miRNA组及空白对照组跨膜细胞数[分别为(52±3)个、(401±28)个及(432±28)个/高位镜(×200)]比较,差异均有统计学意义(P<0.01),而转染非特异性miRNA组与空白对照组之间差异无统计学意义(P>0.05)。重组细胞基底膜(Matrigel)侵袭实验显示,以上3组穿透基底膜细胞数[分别为(48±9)个、(386±26)个及(424±25)个/高位镜(×200)]比较,差异均有统计学意义(P<0.01),而后两组之间无统计学意义(P>0.05)。结论:抑制卵巢癌SKOV3细胞RhoC蛋白的表达对卵巢癌细胞的运动及侵袭有抑制作用,这可能为卵巢癌转移的基因治疗提供新的靶点。

Objective：To investigate the inhibitory effects of RNA interference （RNAi） targeting Ras homologyC（RhoC） on the invasion of ovary carcinoma cell lines SKOV3 in vitro. Methods： RhoC - micro interfering RNAs（miRNA） were designed and the related vectors were constructed by lnvitrogenis RNAi Designer. The vectors were transfected into human ovarian cancer cells SKOV3 through Lipofectamine. Blasticdin was used to select the hardiness single clonies after stable transfection of RhoC miRNA. Human ovarian cancer cells SKOV3 were cultured under being divided into 3 groups： PhoC miRNA group （transfected with RhoC miRNA）, non-specific miRNA group（transfected with non-specific miRNA）, and control group（ without transfection）. The protein expression of Phoc of 3 groups of cells were examined by Western blot, and the inhibitory effects of RhoC miRNA on the movement and invasiveness of ovarian cancer cells were detected by Transwell motility as- say and Matrigel invasion ashy. Results： After SKOV3 cells were stablely transfected, the protein expression of RhoC in RhoC miRNA group [0.198 9（4 257/21 498）1 had greater statistical significance （P 〈 0.01 ） compared with those of the non-specific miRNA group[2. 142（38 245/17 855）] and the control group[2. 604（48 872/18 768）]. It was showed hy the motility activity that the cells infiltrated into Basement Membrane on PhoC miRNA group （52 ± 3）had statistical siguificance （ P 〈 0.01 ）compared with those of the non-specific miRNA group（401 ± 28）and the control group （432±28）. By the invasion activity it was shown the cells were infiltrated into Basement Membrane in RhoC miRNA group （48 ± 9）, which was statistically significant （ P 〈 0.01 ） compared with those of the non-specific miRNA group（ 386 ± 26） and the control group （424 ± 25）. Conclusion： The data indicate that the expressing inhibition of PhoC protein in ovarian cancer cells has supressing effects on the movement and invasiveness of ovarian cancer ceils. RhoC miRNA is possibly a potential target for gene therapy in blocking invasion and metastasis of human ovarian cancer.