RNA干扰技术沉默MCM7基因对人肝癌SMMC-7721细胞生物学行为的影响

Effects of RNA interferencemediated MCM7 knockdown on biological behavior of human liver cancer SMMC-7721 cells

目的:探讨微小染色体维持蛋白7(minichromosome maintenance protein 7,MCM7)基因沉默后对人肝癌细胞系SMMC-7721增殖和凋亡的影响及可能作用机制.方法:利用RNA干扰技术,构建4个靶向MCM7基因shRNA载体(MCM7-shRNA表达载体),并对不同靶点进行有效筛选.将人肝癌细胞系SMMC-7721接种于六孔板,分为3组:以MCM7基因沉默重组慢病毒颗粒(LVshRNA-MCM7)感染SMMC-7721,作为实验组;以对照慢病毒颗粒(LV-shRNA-NC)感染SMMC-7721,作为阴性对照组;空白对照组常规培养,不做任何处理.应用RT-PCR、实时荧光定量PCR和Western blot技术检测MCM7基因mRNA和蛋白的表达,评价干扰效果并筛选有效靶点;MTT法检测细胞体外增生能力,Giemsa染色法检测各组细胞的克隆形成;流式细胞技术(flow cytometry,FCM)分别检测各组细胞增殖和凋亡的变化情况.结果:成功构建MCM7-shRNA表达载体,经测序验证无误,感染SMMC-7721细胞后,细胞荧光显示感染率>90%,内源性靶点得到确认.Western blot及Real-time PCR结果显示:实验组的四种靶点MCM7-shRNA干扰序列中,与阴性对照组和空白对照组比较,MCM7 mRNA和MCM7蛋白的表达水平下调均达50%以上,其中以LV-shRNA-MCM7(4)靶点敲减效率最高,分别达88.95%、87.89%和82.25%、81.63%,差异具有统计学意义(P<0.05),以此作为实验组.MTT法结果显示:实验组细胞490 nm处的吸光度(A)值在转染后24、48、72和96 h时均低于阴性对照组、空白对照组,差别显著(P<0.05).同时Giemsa染色法结果显示:LV-shRNA-MCM7组的克隆形成率(6.00%±0.50%)明显低于空白对照组(14.10%±0.36%)、阴性对照组(13.73%±0.17%),实验组细胞生长明显受到抑制(P<0.05).流式细胞技术显示:实验组较阴性对照组、空白对照组G1期延长,S期缩短,差别显著(P<0.05).实验组细胞凋亡率为(22.27%±1.22%),明显高于阴性对照组(0.05%±0.07%)和空白对照组(0.03%±0.06%),实验组较阴性对照组、空白对照组出现了明显的细胞凋亡(P<0.05).结论:MCM7基因的RNAi重组体可以有效地抑制MCM7基因的表达,RNA干扰技术沉默MCM7基因能够抑制肝癌细胞的生长,阻滞细胞期于G1期,促进其凋亡.

METHODS： Four vectors carrying shRNAs tar- geting the MCM7 gene （MCM7-shRNA expres- sion vector） were constructed and were selected for effective targets. SMMC-7721 cells were di- vided into three groups- an experimental group, a normal control group and a negative control group. The experimental group was transfected with the recombinant lentivirirus vector （LV- shRNA-MCM7）, the negative control was trans- fected with an control lentiviral vector （LV- shRNA-NC）, and the normal control received no treatment. The mRNA and protein levels of MCM7 were analyzed by RT-PCR, quantitative real-time PCR （qPCR）, and Western blot. Cell proliferation was detected by MTT assay, and cell colony formation was detected by Giemsa staining. Cell cycle progression and apoptosis were observed by flow cytometry （FCM）. RESULTS： MCM7-shRNA expression vectors were successfully constructed and verified by DNA sequencing. After transfecting SMMC-7721 cells with various vectors, cell fluorescence was observed in 〉 90% of cells. MCM7 mRNA and protein expression in the four MCM7-shRNA groups was down-regulated by 〉 50% compared with the negative control group and normal con- trol group. The LV-shRNA-MCM7 vector had the highest efficiency and was used in subse- quent experiments. MTT results showed that cell proliferation in the experimental group at 24, 48, 72 and 96 h after transfection was significantly lower than that in the negative control group and normal control group （P 〈 0.05 for all）. Gi- emsa staining results showed that the colony formation rate was significantly lower in the ex- perimental group than in the two control groups （6.00% ± 0.50% vs 14.10% ± 0.36%, 13.73% ± 0.17%, P 〈 0.05 for both）. FCM analysis showed that the percentage of cells in G1 phase increased in cells transfected with the MCM7-shRNA （P 〈 0.05 for both）. The apoptosis rate was significantly higher in the experimental group than in the negative control group and blank control group （22.27% ± 1.22% vs 0.05% ± 0.07%, 0.03% ± 0.06%, P 〈 0.05 for both）.CONCLUSION： RNAi-induced MCM7 down- regulation could inhibit cell growth, suppress cell colony formation, block the cell cycle at G1 phase, and induce cell apoptosis in SMMC-7721 cells.