DNA甲基转移酶1基因沉默对HT-29细胞凋亡的影响

Effects of knock-down of DNMT1 gene on apoptosis in HT-29 cells

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。

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,P0.001）.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,P0.01）.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,P0.001）.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 （P0.05）.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.