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目的观察表达短发夹状RNA(shRNA)的mU6/survivin质粒转染卵巢癌细胞株OVCAR3细胞后,对OVCAR3细胞survivin mRNA表达的抑制作用,以及对OVCAR3细胞紫杉醇药物敏感性的影响。方法将表达绿色荧光蛋白的pEGFPC2质粒与脂质体按不同比例转染OVCAR3细胞,流式细胞仪检测其转染效率。根据转染效率最高时pEGFPC2质粒与脂质体的比例,将mU6/survivin质粒转染入OVCAR3细胞。实验分为3组未转染组、脂质体组、转染组,RT-PCR技术检测3组OVCAR3细胞中survivin mRNA的表达,流式细胞仪分析细胞周期及细胞凋亡率的变化。四甲基偶氮唑蓝(MTT)比色法检测3组OVCAR3细胞对紫杉醇的50%抑制浓度(IC50)。结果pEGFPC2质粒与脂质体按1∶2比例转染OVCAR3细胞时其转染效率最高,达23.6%。将mU6/survivin质粒与脂质体也按1∶2比例转染OVCAR3细胞24h后,未转染组、脂质体组、转染组细胞survivin mRNA的相对含量分别为0.81±0.05、0.79±0.12、0.26±0.04,转染组survivin mRNA相对含量下降至未转染组的32%,两组比较,差异有统计学意义(P<0.01)。转染组转染24h后,细胞凋亡率为(31.9±1.2)%,明显高于未转染组的(4.9±0.7)%和脂质体组的(5.6±0.5)%(P=0.000);转染组细胞周期被阻滞于G0/G1期,G2/M期减少,分别与未转染组比较,差异有统计学意义(P<0.01)。MTT比色法检测结果显示,未转染组、脂质体组、转染组OVCAR3细胞对紫杉醇的IC50分别为(0.305±0.032)、(0.157±0.031)、(0.019±0.001)μmol/L,转染组OVCAR3细胞对紫杉醇的IC50降低为未转染组的1/16,两组比较,差异有统计学意义(P=0.000)。结论shRNA可有效抑制卵巢癌细胞survivinmRNA的表达,并显著提高了卵巢癌细胞对紫杉醇的药物敏感性。
Objective To investigate the inhibitory effect of mU6 / survivin plasmid transfected ovarian cancer cell line OVCAR3 expressing short hairpin RNA (shRNA) on the expression of survivin mRNA in OVCAR3 cells and its effect on paclitaxel sensitivity in OVCAR3 cells. Methods pEGFPC2 plasmid and liposome were transfected into OVCAR3 cells with different ratios. The transfection efficiency was detected by flow cytometry. According to the ratio of pEGFPC2 plasmid to liposome at the highest transfection efficiency, mU6 / survivin plasmid was transfected into OVCAR3 cells. The experiment was divided into three groups of untransfected group, liposome group, transfection group, RT-PCR technology to detect the expression of survivin mRNA in the three groups of OVCAR3 cells, flow cytometry analysis of cell cycle and apoptosis rate changes. The 50% inhibitory concentration (IC50) of paclitaxel in three groups of OVCAR3 cells was determined by MTT assay. Results The transfection efficiency of pEGFPC2 plasmid and liposome at the ratio of 1: 2 was the highest, reaching 23.6%. After transfection of mU6 / survivin plasmid and liposome into OVCAR3 cells at a ratio of 1: 2 for 24 hours, the relative contents of survivin mRNA in untransfected, lipofectamine and transfected cells were 0.81 ± 0.05 and 0.79 ± 0.12 , 0.26 ± 0.04. The relative content of survivin mRNA in transfected group decreased to 32% of that in untransfected group, the difference was statistically significant (P <0.01). After transfection for 24 h, the apoptotic rate was (31.9 ± 1.2)%, which was significantly higher than that in the untransfected group (4.9 ± 0.7%) and the liposome group (5.6 ± 0.5)% ). The cell cycle was blocked in G0 / G1 phase and G2 / M phase in transfected group, respectively. Compared with untransfected group, the difference was statistically significant (P <0.01). The results of MTT assay showed that IC50 of OVCAR3 cells in untransfected, liposome and transfected groups were (0.305 ± 0.032), (0.157 ± 0.031), (0.019 ± 0.001) μmol / L, The IC50 of paclitaxel in OVCAR3 cells in transfection group was 1/16 lower than that in non-transfected cells, the difference was statistically significant (P = 0.000). Conclusion shRNA can effectively inhibit the expression of survivin mRNA in ovarian cancer cells and significantly increase the drug sensitivity of ovarian cancer cells to paclitaxel.