pshRNA-DNMT1介导的胃癌AGS细胞凋亡的体内外研究

目的构建沉默DNMT1基因的重组体pshRNA-DNMT1,研究其体内外对胃癌AGS细胞增殖凋亡的影响.方法设计短发夹RNA（shRNA）的寡核苷酸片断,克隆到载体pTZU6＋1中,构建重组体pshRNA-DNMT1,转染胃癌细胞株AGS,用Western 印迹检测DNMT1蛋白质水平变化,用逆转录-聚合酶链反应（RT-PCR）法评估mRNA水平,四甲基偶氮唑盐比色法（MTT法）动态监测活细胞数,电镜和原位末端标记技术（TUNEL）观察其体外的促凋亡作用.构建裸鼠胃癌模型,经pshRNA-DNMT1处理后观察瘤体大小,绘制生长曲线;用苏木素伊红（HE）染色法、电镜和TUNEL检测细胞凋亡,用增殖细胞核抗原（PCNA）法评价细胞的增殖能力.结果成功构建重组质粒pshRNA-DNMT1后,进行序列分析得到确证.pshRNA-DNMT1转染AGS细胞后24 h出现DNMT1蛋白质表达量减少,抑制率为28.24%,48 h为68.54%,72 h为81.47%,重组质粒pshRNA-DNMT1对胃癌AGS细胞中DNMT1基因的转录有着明显抑制作用,转染后24 h抑制率为21.63%,48 h为52.97%,72 h为72.06%.转染后24、48和72 h细胞数存活率为PBS组的79.49%、51.63%和39.16%.pshRNA-DNMT1能抑制肿瘤生长,呈一定的时间和剂量依赖性.结论重组质粒pshRNA-DNMT1能特异有效地抑制胃癌细胞株AGS内DNMT1基因的表达.在胃癌细胞株AGS的体内外实验中均能抑制细胞增殖,促进细胞凋亡.

Effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer: experiments in vitro and in vivo

OBJECTIVE: To investigate the effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer. METHODS: Recombinant eukaryotic expression plasmid pshRNA-DNMT1 containing the sequence of the gene of DMNT1 that methylates the specific pyrimidine residue in the DNA promoter region was constructed. Human gastric cells of the line AGS were cultured and transfected with pshRNA-DNMT1. Western blotting was used to detect the protein expression of DNMT1 of the AGS cells, and RT-PCR was used to detect the mRNA expression of DNMT1 of the AGS cells. MTT method was used to dynamically monitor the surviving cells and the cell apoptotic was observed by electron microscopy and TUNEL method. Forty nude the mice were inoculated with suspension of AGS cells. When the tumor reached the size of 5 - 6 mm in diameter the mice were randomly divided into 5 equal groups to be injected intravenously with PBS, liposome, pTZU6 + 1, pshRNA-DNMT1 of medium dose, and pshRNA-DNBMT1 of large dose for 4 times with an interval of 3 days. The tumor size was measured every day. Three days after the last injection the mice were killed and the tumors were taken out to undergo light and electron microscopy and TUNEL method to detect the cell apoptosis. Immunohistochemistry was used to detect the proliferating cell nuclear antigen (PCNA) of the cells. RESULTS: The protein and mRNA expression levels of DNMT1 in the cultured AGS cells 24, 48, and 72 hours after transfection of the pshRNA-DNMT1 group were all lower than those of the control group. The numbers of surviving AGS cells of the pshRNA-DNMT1 group became significantly gradually lower than those of the liposome and pTZU6 + 1 groups since 24 hours after transfection (all P < 0.05). The apoptotic rate of AGS cells in the pshRNA-DNMT1 group was 34.78% +/- 0.52%, significantly higher than those of the liposome and pTZU6 + 1 groups (4.86% +/- 0.17% and 5.12% +/- 0.76% respectively, both P < 0.05). The subcutaneous tumors of the mice of the PGS, liposome, and pTZU6 + 1 groups augmented along with time without significant differences among these 3 groups (all P > 0.05). The tumor of the 2 pshDNMT1 groups began to augment since the 5(th) day and began to be reduced in size since the 10(th) day in comparison with the other 3 groups (all P < 0.05), and the tumor size of the pshRNA-DNMT1 (large dose) group was significantly smaller than that of the pshRNA-DNMT1 (medium dose) group 15 days after the injection (P < 0.05). The rates of cell apoptosis of the pshRNA-DNMT1 (large dose) and pshRNA-DNMT1 (medium dose) groups were both significantly higher than those of the other 3 groups (all P < 0.05) and with a sufficient difference between these 2 pshRNA-DNMT1 groups (P < 0.05). PCNA analysis showed that the proliferation activity of the cells in the pshRNA-DNMT1 groups was significantly suppressed. CONCLUSION: The recombinant plasmid pshRNA-NMT1 effectively and specifically inhibits the expression of the gene DNMT1, thus inhibiting the proliferation and inducing the apoptosis of gastric cancer cells.