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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 effect of RNA interference (RNAi) down-regulation of RhoA expression on the malignant phenotype of ovarian cancer cell line SKOV-3. Methods: The eukaryotic expression vector of short hairpin RNA (shRNA) of RhoA gene was constructed and transfected into ovarian cancer cell line SKOV-3 by lipofectamine. The expression of RhoA in SKOV-3 after RNAi was detected by RT-PCR and Western blotting MRNA and protein expression levels were observed in Boyden chamber. Invasion and migration of Boyden chamber were observed in vitro and cell invasion and migration were observed. MTT assay was used to determine the changes of cell proliferation and adhesive ability before and after transfection. Results: Compared with control cells transfected with RhoA shRNA, the expression of RhoA mRNA was (5.46 ± 2.02)% and (56.37 ± 17.42)%, respectively, and the protein expression was (6.03 ± 2.04 ) And (59.03 ± 19.94)%, respectively. The mean percentage of cell invasion was (15.84 ± 4.17)% and (33.64 ± 4.64)%, and the percentage of healing was (44.79 ± 6.21) and (68.36 ± 4.46) Values were 0.726 ± 0.166 and 1.703 ± 0.340 respectively, P <0.05 (n = 3), while the adhesion values (A values) were 0.833 ± 0.137 and 0.894 ± 0.189, respectively. Conclusion: The decrease of RhoA expression by RNAi can reduce the invasion, migration and proliferation of ovarian cancer cell line SKOV-3 in vitro.