沉默DNA甲基转移酶1基因对子宫颈癌细胞增殖及凋亡的影响

目的 利用RNA干扰技术沉默宫颈癌HeLa细胞中DNA甲基转移酶(DNMT)1基因的表达,探讨沉默DNMT1基因后对HeLa细胞增殖及凋亡的影响.方法 针对DNMT1基因构建短发夹状RNA(shRNA)重组载体pshRNA-DNMT1-A、B和C质粒,并行酶切鉴定和测序分析.采用RT-PCR技术筛选出其中RNA干扰效果最佳的重组载体,通过脂质体介导转染宫颈癌HeLa细胞(转染组),以空载体组(转染空载体pSilencer3.1-H1质粒)和未转染组(未作任何处理)为对照.采用RT-PCR技术和蛋白印迹法分别检测转染前后HeLa细胞中DNMT1 mRNA和蛋白表达的变化,活细胞计数(CCK-8)法和双染法流式细胞术分别检测转染前后HeLa细胞增殖和凋亡的变化.结果 (1)酶切鉴定和测序分析证实,靶向DNMTI的3个shRNA重组载体pshRNA-DNMT1-A、B和C质粒构建成功.RT-PCR技术筛选显示,重组载体pshRNA-DNMT1-B质粒的干扰效果最佳.(2)RT-PCR技术检测显示,转染组转染24、48及72 h时HeLa细胞中DNMT1 mRNA的相对表达水平分别为0.406±0.057、0.191±0.036、0.104±0.015,明显低于空载体组和未转染组(分别为0.520±0.020、0.537±0.041,P＜0.05).蛋白印迹法检测显示,转染组转染24、48及72 h时细胞中DNMT1蛋白的相对表达水平分别为0.197±0.024、0.075±0.015、0.040±0.013,明显低于卒载体组和未转染组(分别为0.273±0.010、0.283±0.016,P＜0.05).(3)CCK-8法检测显示,转染组转染24、48、72、96、120 h时HeLa细胞存活率分别为70.8%、64.8%、51.6%、45.3%、38.0%,分别与空载体组和未转染组各时间点比较,差异均有统计学意义(P＜0.01).双染法流式细胞术检测显示,转染组转染24、48及72 h时的细胞凋亡率分别为(17.7±1.3)%、(35.3±1.3)%、(47.6±1.6)%,明显高于空载体组及未转染组[分别为(4.9±0.5)%、(5.1±0.7)%,P＜0.05].结论 RNA干扰技术能成功沉默宫颈癌HeLa细胞中DNMT1基因的表达,通过下调DNMT1基因的表达可抑制宫颈癌细胞增殖、促进细胞凋亡.

Effect of DNA methyltransferase 1 gene silencing on proliferation and apoptosis of HeLa cells

Objective To observe the effect of DNA methyltransferase 1 (DNMT1 ) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. Methods Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3.1-HI and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8 ) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry(FCM ) method. Results Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector PShRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMTI-B was the better choice. While no effect of pshRNA-DNMTI-C was seen. BT-PCR results showed that the relative mRNA expression of DNMT1 gene in Helm cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406±0.057,0.191±0.036 and 0.104±0.015, which were significantly lower than that in Helm cells transfected by empty vector and non-transfected cells (0.520±0.020, 0.537±0.041, respectively, P ＜ 0.05 ). The western blotting analysis manifested that the relative expression of DNMT1 protein of Helm cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197±0.024, 0.075±0.015, 0.040± 0.013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273±0.010, 0.283±0.016, respectively, P ＜0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P ＜ 0.01 ). The results of FCM indicated that the apoptesis rate of HeLa cells trandected with pshRNA-DNMTI for 24, 48 and 72 hours were (17.7± 1.3 ) %, (35.3±1.3 ) %, (47.6±1.6 ) %, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9±0.5 ) %, (5.1±0.7 ) %, respectively, P ＜ 0.05]. Conclusions DNMT1 can be successfully silenced by RNA interfering in cervical Helm cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.