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。
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（P0.05,n=3）,and the value of adhesion was 0.833±0.137 and 0.894±0.189 （P0.05,n=3）. CONCLUSION：RNAi-induced RhoA gene silencing may decrease the capacity of SKOV-3 invasion,migration and proliferation in vitro.
出处
《中华肿瘤防治杂志》
CAS
2010年第17期1321-1324,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
国家重点基础研究发展规划973项目(2009CB521800)
