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目的:观察RNA干扰(RNAi)技术对卵巢上皮癌HO8910细胞株RhoA基因表达的抑制作用,探讨其对HO8910细胞侵袭、迁移的影响。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HO8910,实时荧光定量PCR和蛋白质印迹法检测RNAi后HO8910中RhoA的mRNA及蛋白表达水平变化,Transwell小室体外侵袭和划痕试验检测细胞侵袭和迁移能力。结果:转染RhoA shRNA(质粒1、2和3)组的RhoA mRNA的表达明显弱于未转染任何质粒的空白组和对照组,P<0.05,且质粒2组表达最低(27.9%),抑制效率最高(72.1%),具有序列特异性。转染序列特异性RhoA shRNA的细胞(实验组)与对照组比较,RhoA蛋白的表达分别为(6.08±2.24)%和(59.25±17.96)%,P<0.05;细胞平均侵袭能力测值为18.82±2.61和35.25±5.12,P<0.05;愈合百分比为(43.80±6.21)%和(69.35±4.42)%,P<0.05。结论:靶向RhoA的shRNA可抑制卵巢癌HO8910细胞RhoA基因的表达,能降低细胞体外侵袭和迁移能力。
OBJECTIVE: To observe the inhibitory effect of RNA interference (RNAi) technology on RhoA gene expression in HO8910 epithelial ovarian cancer cell line, and to investigate its effect on invasion and migration of HO8910 cells. Methods: Eukaryotic expression vector of short hairpin RNA (shRNA) of RhoA gene was constructed. The expression of RhoA mRNA and protein in HO8910 after RNAi were detected by real-time fluorescence quantitative PCR and Western blotting using liposome-mediated transfection of ovarian cancer HO8910 cells. Protein expression level changes, Transwell chamber in vitro invasion and scratch test to detect cell invasion and migration. Results: The expression of RhoA mRNA in transfected RhoA shRNA (plasmids 1, 2 and 3) group was significantly weaker than that in the control group and untransfected plasmids (P <0.05). The expression of RhoA mRNA in plasmids 2 and 3 group was the lowest (27.9% The highest inhibition efficiency (72.1%), with sequence specificity. Compared with control group, the expression of RhoA protein was (6.08 ± 2.24)% and (59.25 ± 17.96)%, respectively, in cells transfected with sequence-specific RhoA shRNA (P <0.05). The mean invasiveness of cells was 18.82 ± 2.61 and 35.25 ± 5.12, P <0.05; the percentages of healing were (43.80 ± 6.21)% and (69.35 ± 4.42)%, respectively, P <0.05. Conclusion: shRNA targeting RhoA can inhibit the expression of RhoA gene in ovarian cancer cell line HO8910 and reduce the invasion and migration of cells in vitro.