【摘要】 目的:观察DNA甲基转移酶1(DNMT1)基因沉默对结肠癌HT-29细胞凋亡的影响。方法:HT-29细胞分为3组:未处理组、对照siRNA组及DNMT1siRNA组,后2细胞转染对照siRNA和DNMT1siRNA,24h后,通过RT-PCR和Westernblot观察3组细胞DNMT1、Caspase-3/9、bcl-2及BaxmRNA和蛋白表达的变化,通过流式细胞术分析HT-29细胞凋亡,并测定Caspase-3/9的活性。结果:DNMT1siRNA组中DNMT1mRNA和蛋白的相对表达量为(0.273±0.016)和(1.127±0.019),低于未处理组[分别为(1.729±0.023)和(2.741±0.031)]和对照siRNA组[分别为(1.751±0.018)和(2.673±0.027)](F分别为5822.683和3656.560,P均<0.001)。DNMT1siRNA组细胞凋亡率为(22.65%±1.67%),高于未处理组(7.92%±1.29%)和对照siRNA组(8.13%±1.21%)(F=108.460,P<0.001)。DNMT1siRNA组细胞的Caspase-3/9活性[分别为(2671.83±57.63)和(2187.71±68.35)]高于未处理组[(196.47±16.73)和(511.35±34.90)]和对照siRNA组[(215.29±14.38)和(534.78±41.70)](F分别为4790.975和1089.891,P均<0.001)。与未处理组和对照组相比DNMT1siRNA组Bcl-2mR-NA和蛋白的表达降低,但Caspase-3/9和BaxmRNA和蛋白的表达升高(P<0.05)。结论:DNMT1的下调能诱导HT-29细胞凋亡,其可能的作用机制与caspase-3/9、bcl-2和bax表达的变化密切相关。更多还原

【Abstract】 Aim:To investigate the effects of knock-down of DNMT1 gene on cell apoptosis of colon carcinoma cell line HT-29.Methods:Cells were divided into three groups:untreated group,control siRNA group and DNMT1 siRNA group,and the two latter transfected control siRNA or DNMT1 siRNA by Lipofectamine 2000.Cell apoptosis was investigated by Flow cytometry,and caspase-3/9 activities was analyzed,and expressions of DNMT1,Caspase-3/9,bcl-2 and bax mRNAs and proteins were detected using semi-quantitative RT-PCR and Western blotting methods.Results:Expressions of DNMT1 mRNA and protein in DNMT1 siRNA group [(0.273±0.016) and (1.127±0.019)] were obviously lower than that in untreated group [(1.729±0.023) and (2.741±0.031)] and control siRNA group [(1.751±0.018) and (2.673±0.027)] (F=5 822.683 and 3 656.560,P<0.001).Apoptosis rate in DNMT1 siRNA group (22.65%±1.67%) was higher than those in untreated group (7.92%±1.29%) and control siRNA group (8.13%±1.21%) (F=108.460,P<0.01).Compared to untreated group [(196.47±16.73) and (511.35±34.90)] and control siRNA group [(215.29±14.38 and 534.78±41.70)],Caspase-3/9 activities [(2 671.83±57.63) and (2 187.70±68.35)] were significantly improved in DNMT1 siRNA group (F=4 790.975 and 1 089.891,P<0.001).Further,expression of bcl-2 mRNA and protein was evidently inhibited,whereas expressions of caspase-3/9 and bax mRNAs and proteins were evidently enhanced after transfection with DNMT1 siRNA for 24 h (P<0.05).Conclusion:Down-regulation of DNMT1 could induce cell apoptosis in HT-29 cells,which is associated with the changes of apoptosis related caspase-3/9,bcl-2,and bax expression.更多还原