Zhao Xian-lan, Rao Yan-ling, Qiao Yu-huan, Zhang Hui-li
Department of Obstetrics and Gynecology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China.
Zhonghua Fu Chan Ke Za Zhi. 2009 Jul;44(7):533-7.
To observe the effect of DNA methyltransferase 1 (DNMT1) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells.
Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3. 1-H1 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.
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-DNMT1-B was the better choice. While no effect of pshRNA-DNMT1-C was seen. RT-PCR results showed that the relative mRNA expression of DNMT1 gene in HeLa 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 HeLa 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 HeLa 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 apoptosis rate of HeLa cells transfected with pshRNA-DNMT1 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].
DNMT1 can be successfully silenced by RNA interfering in cervical HeLa cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.
观察RNA干扰技术沉默DNA甲基转移酶1（DNMT1）基因对HeLa细胞增殖和凋亡的影响。
采用脂质体2000分别将重组质粒pshRNA-DNMT1-A、B和C转染入HeLa细胞，同时将转染质粒载体pSilencer3.1-H1的细胞及未处理的细胞作为对照组。采用RT-PCR筛选抑制效果最佳的重组质粒。运用RT-PCR和蛋白质免疫印迹法检测转染24、48和72小时后HeLa细胞中DNMT1的mRNA和蛋白表达。采用细胞计数试剂盒-8（CCK-8）法检测转染后HeLa细胞的增殖情况，通过流式细胞术（FCM）检测细胞凋亡情况。
成功将3条靶向DNMT1的短发夹RNA（shRNA）A、B和C插入质粒载体pshRNA，所获shRNA的编码序列与设计片段一致。结果表明，重组质粒pshRNA-DNMT1-A和B均可有效敲低人宫颈癌细胞中DNMT1基因的表达，其中pshRNA-DNMT1-B效果更佳，而pshRNA-DNMT1-C无明显作用。RT-PCR结果显示，转染pshRNA-DNMT1 24、48和72小时后，HeLa细胞中DNMT1基因的相对mRNA表达分别为0.406±0.057、0.191±0.036和0.104±0.015，显著低于空载体转染组和未转染组（分别为0.520±0.020、0.537±0.041，P<0.05）。蛋白质免疫印迹分析表明，转染pshRNA-DNMT1 24、48和72小时后，HeLa细胞中DNMT1蛋白的相对表达分别为0.197±0.024、0.075±0.015、0.040±0.013，显著低于空载体转染组和未转染组（分别为0.273±0.010、0.283±0.016，P<0.05）。CCK-8结果显示，转染pshRNA-DNMT1 24、48、72、96和120小时后，HeLa细胞的存活率分别为70.8%、64.8%、51.6%、45.3%和38.0%，与空载体转染组和未转染组在不同时间点比较，差异均有统计学意义（P<0.01）。FCM结果表明，转染pshRNA-DNMT1 24、48和72小时后，HeLa细胞的凋亡率分别为（17.7±1.3）%、（35.3±1.3）%、（47.6±1.6）%，显著高于空载体转染组和未转染组[分别为（4.9±0.5）%、（5.1±0.7）%，P<0.05]。
RNA干扰可成功沉默宫颈HeLa细胞中的DNMT1基因。下调DNMT1可抑制宫颈癌细胞增殖并诱导细胞凋亡。
