【摘要】 目的:探讨RNA干扰(RNAi)下调RhoA表达对卵巢癌细胞SKOV-3恶性表型的影响。方法:构建RhoA基因特异性短发夹状RNA(shRNA)真核表达载体,以脂质体介导转染卵巢癌细胞SKOV-3,RT-PCR和蛋白质印迹法检测RNAi后SKOV-3中RhoA的mRNA及蛋白表达水平变化;Boyden小室体外侵袭和划痕实验观察细胞侵袭和迁移能力,MTT比色法测定转染前后细胞增殖及粘附能力的变化。结果:转染RhoA shRNA的实验组细胞与转染空载体的对照组细胞比较,RhoA mRNA的表达分别为(5.46±2.02)%和(56.37±17.42)%,蛋白质的表达分别为(6.03±2.04)%和(59.03±19.94)%,细胞平均侵袭百分比为(15.84±4.17)%和(33.64±4.64)%,愈合百分比为(44.79±6.21)%和(68.36±4.46)%,增殖能力(A值)分别为0.726±0.166和1.703±0.340,P<0.05(n=3);而粘附能力(A值)分别为0.833±0.137和0.894±0.189,P>0.05(n=3)。结论:通过RNAi降低RhoA的表达能降低卵巢癌细胞SKOV-3体外侵袭、迁移和增殖能力。更多还原

【Abstract】 OBJECTIVE:To investigate the effects of RhoA gene silencing induced by RNA interference (RNAi) on SKOV-3 cell malignant behaviors in vitro. METHODS:The vector containing shRNA targeting RhoA gene was constructed and transfected into human ovarian carcinoma cell line SKOV-3. The expressions of mRNA and protein in SKOV-3 were detected respectively by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Boyden chamber in vitro invasion assay and wound-healing assay were used to evaluate the invasive and migratory capability of SKOV-3. The changes in the adhesion and proliferation of the cells were detected by MTT assay. RESULTS:The group transfected with RhoA shRNA was compared with the control group. The expression of RhoA mRNA was (5.46±2.02)% and (56.37±17.42)%. The protein level was (6.03±2.04)% and (59.03±19.94)%. The cell invasion percentage was (15.84±4.17)% and (33.64±4.64)%. The percent wound closure was (44.79±6.21)% and (68.36±4.46)%. The absorbance value of proliferation was 0.726±0.166 and 1.703±0.340(P<0.05,n=3),and the value of adhesion was 0.833±0.137 and 0.894±0.189 (P>0.05,n=3). CONCLUSION:RNAi-induced RhoA gene silencing may decrease the capacity of SKOV-3 invasion,migration and proliferation in vitro.更多还原