论文部分内容阅读
目的:建立慢病毒介导RhoA基因稳定沉默卵巢癌HO8910细胞株,研究RhoA基因对HO8910细胞侵袭和迁移的影响。方法:构建靶向沉默RhoA基因的慢病毒载体Lenti-U6-shRhoA,将其感染HO8910细胞,经过嘌呤霉素筛选,建立稳定沉默RhoA基因的HO8910细胞。实时荧光定量PCR检测HO8910细胞中RhoA mRNA的表达;MTT检测HO8910细胞的增殖;Transwell体外迁移和侵袭实验检测RhoA基因沉默对HO8910细胞侵袭和迁移的影响。结果:成功构建靶向RhoA基因的慢病毒载体Lenti-U6-shRhoA,建立了RhoA基因稳定沉默的HO8910细胞。与Lenti-U6-NC对照组对比,RhoA基因稳定沉默HO8910细胞RhoA mRNA的表达明显降低[(30.7±5.40)%vs(95.9±6.53)%,P<0.05];沉默RhoA基因能降低HO8910细胞的存活率[(51.16±7.41)%vs(95.67±2.14)%,P<0.05];RhoA基因沉默组HO8910细胞的侵袭、迁移及黏附能力较Lenti-U6-NC对照组明显受到抑制[迁移穿过人工基底膜细胞数(31±6)vs(84±13)个,P<0.05;穿过微孔膜的细胞数(97±12)vs(689±23)个,P<0.05;黏附率(68.16±6.53)%vs(98.71±4.92)%,P<0.05]。结论:慢病毒介导的RhoA基因沉默能抑制卵巢癌HO8910细胞的侵袭和迁移。
OBJECTIVE: To establish a lentivirus-mediated RhoA gene silencing HO8910 cell line and to investigate the effect of RhoA gene on the invasion and migration of HO8910 cells. Methods: Lenti-U6-shRhoA, a lentiviral vector targeting silencing of RhoA gene, was constructed and transfected into HO8910 cells. After puromycin screening, HO8910 cells stably silenced by RhoA gene were constructed. The expression of RhoA mRNA in HO8910 cells was detected by real-time fluorescence quantitative PCR. The proliferation of HO8910 cells was detected by MTT assay. The invasion and migration of HO8910 cells were detected by Transwell in vitro migration and invasion assay. Results: The lentiviral vector Lenti-U6-shRhoA targeting RhoA gene was successfully constructed and the HO8910 cell with stable RhoA gene was established. Compared with Lenti-U6-NC control group, RhoA gene expression was significantly decreased in HO8910 cells [(30.7 ± 5.40)% vs (95.9 ± 6.53)%, P <0.05] (51.16 ± 7.41)% vs (95.67 ± 2.14)%, P <0.05]. The invasion, migration and adhesion of HO8910 cells in RhoA gene silencing group were significantly inhibited compared with Lenti-U6-NC control group The number of cells in the artificial basement membrane was (31 ± 6) vs (84 ± 13), P <0.05. The number of cells passing through the microporous membrane was (97 ± 12) vs (689 ± 23), P < 68.16 ± 6.53)% vs (98.71 ± 4.92)%, P <0.05]. Conclusion: Lentivirus-mediated RhoA gene silencing can inhibit the invasion and migration of ovarian cancer cells HO8910.