Dnmt1基因对胃癌细胞株AGS细胞增殖凋亡的影响

目的:利用RNA干扰(RNAi)技术,以Dnmt1(DNA methyltransferase 1)为靶基因,设计构建重组体pshRNA-Dnmt1,研究其对胃癌AGS细胞增殖凋亡的影响.方法:设计shRNA的寡核苷酸片断,再克隆至载体pTZU6 1中构建重组体pshRNA-Dnmt1,转染胃癌细胞株AGS,用Western Blot检测DNMT1蛋白水平变化,用RT-PCR法评估mRNA水平,MTT法动态监测活细胞数,AO/EB法、电镜和TUNEL观察其促凋亡作用.结果:成功构建重组质粒pshRNA-Dnmt1 后,进行序列分析得到确证. RT-PCR检测结果证实: 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抑制率在21.63%左右;48 h为52.97%;72 h为72.06%. Western Blot检测结果表明:pshRNA-Dnmt1 转染AGS细胞后24 h出现DNMT1蛋白表达量减少,抑制率为28.24%;48 h为68.54%;72 h为81.47%. MTT结果提示: 转染后24, 48和72 h细胞数存活率为对照组的79.49%, 51.63%和39.16%. AO/EB法、电镜和TUNEL都看见大量典型的凋亡和坏死细胞,转染后48 h的细胞凋亡率由对照组的5%上升为35%左右.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,能抑制细胞增殖,促进细胞凋亡,从而为肿瘤的基因治疗开辟了新途径.

Effects of Dnmt1 gene on proliferation and apoptosis of gastric cancer cell line AGS

AIM: To design a recombinant plasmid pshRNA- dnmt1 by RNA interference and to assess its effects on DNA methyltransferase (Dnmt1) gene expression in gastric cancer cell line AGS, and on the proliferation and apoptosis of AGS cells in vitro. METHODS: The eukaryotic expression plasmid pshRNA- Dnmt1, containing the sequence of shRNA, was cloned and transfected into AGS cells. The mRNA levels of gene Dnmt1 were determined by RT-PCR, the protein expression levels by Western Blot, the cell survival index by MTT assay, and the apoptosis was also evaluated by AO/EB, electron microscope and TUNEL. RESULTS: A recombinant plasmid pshRNA-Dnmt1 was successfully constructed and confirmed by sequencing analysis. PshRNA-Dnmt1 induced a 21.63% inhibition of Dnmt1 mRNA after 24 h treatment, 52.97% after 48 h, and 72.06% after 72 h. Dnmt1 protein levels in AGS cells appeared to reduce, with inhibition rate of 28.24% after 24 h of treatment, 68.54% after 48 h, and 81.47% after 72 h. MTT results showed that the cell survival rates of AGS cells after transfected with pshRNA-Dnmt1 at 24 h, 48 h and 72 h respectively were 79.49%, 51.63% and 39.16%. AO/EB, electron microscope and TUNEL test indicated: after transfection with the recombinant plasmid, there existed a lot of apoptotic and necrotic cells, and the apoptosis rate increased from about 5% to 35%. CONCLUSION: The recombinant plasmid pshRNA-Dnmt1 can efficiently and specifically inhibit the expression of gene Dnmt1, inhibit the proliferation of AGS cells, and induce the apoptosis.