去甲基化机制在腺苷及同型半胱氨酸诱导肝癌HepG2细胞凋亡中的作用

Effect of demethylation on adenosine and homocysteine-induced apoptosis in HepG2 cells

目的探索去甲基化机制在腺苷(ADO)及同型半胱氨酸(HCY)诱导肝癌HepG2细胞凋亡中的作用。方法CCK8法检测不同浓度腺苷(1.0、2.0、4.0 mol·L-1)单独或者联合同型半胱氨酸作用肝癌HepG2细胞不同时间(6、12、24 h)后细胞存活率;亦使用DNA甲基化酶抑制剂5-AzaCdR观察其对细胞生长的影响。AnnexinV-FITC/PI双染法检测细胞凋亡率,流式细胞术检测细胞内线粒体膜电位:实时荧光定量PCR及Western免疫印迹法分别检测caspase-3、caspase-8、caspase-9、MDM-2、p53、Cytochrome C、DNMT1、DNMT3a、DNMT3b等因子的表达量。结果 ADO联合HCY明显抑制HepG2细胞增殖,呈剂量和时间依赖性;不同浓度ADO联合HCY(1.0、2.0、4.0 mol·L-1)作用HepG2细胞24h,细胞凋亡率分别为(18.63±1.25)%,(29.42±2.37)%,(42.47±3.09)%,均明显高于阴性对照组(1.30±0.82)%,P<0.01;联合用药组线粒体膜电位明显下降,分别从空白对照组(674.15±82.8)%下降为(428.38±54.5)%、(297.78±30.5)%、(74.45±5.73)%,P<0.01;ADO联合HCY导致caspase-3、caspase-8、caspase-9、p53、Cytochrome C表达上调,MDM-2表达下调。ADO联合HCY或5-Aza-CdR处理HepG2细胞均可抑制DNA(甲基化酶DNMT1、DNMT3a、DNMT3b)mRNA表达量。结论 ADO与HCY联合作用引起了细胞内甲基化改变,低甲基化作用激活了p53及线粒体等凋亡通路,最终导致肝癌HepG2细胞凋亡。

Aim To investigate the mechanism of demethylation on adenosine （ADO ）and homocysteine＆amp;nbsp;（HCY）-induced apoptosis in human hepatoma HepG2 cells .Methods HepG 2 cells were treated with differ-ent concentrations of ADO （1.0、2.0、4.0 mol · L-1 ） alone or in combination with HCY for 6h,12h and 24h,5-aza-2-deoxycytidine （5-Aza-CdR）as a positive control.Cell viabilities were assessed by CCK8 assay. Cell apoptosis was observed by AnnexinV-FITC/PI staining.The mitochondrial membrane potentials（ΔΨ） were measured by flow cytometry.The mRNA and pro-tein expressions of caspase-3,caspase-8,caspase-9, MDM-2,p53,Cytochrome C,DNMT1,DNMT3a,DN-MT3 b were detected by RT-qPCR and Western blot re-spectively.Results ADO alone or in combination with HCY significantly reduced the viability of HepG2 cells in a dose and time-dependent manner.The apoptotic rates of HepG2 cells after combination treatment with ADO and HCY at 1 .0,2.0,4.0 mol · L-1 for 24 h were （1 8.63 ± 1.25 ）%,（29.42 ±2.37 ）% and （42.47 ±3.09 ）%,compared with the control group （1.30 ±0.82 ）%,P ＆lt;0.01;and the mitochondrial＆amp;nbsp;membrane potentials were decreased from 674.15 ± 82.8%（black control group）to （428.38 ±54.5）%, （297.78 ±30.5）%,（74.45 ±5.73）%,P＆lt;0.01, respectively.The expressions of caspase-3,caspase-8, caspase-9,MDM-2,p53,Cytochrome C were up-regula-ted and MDM-2 were down-regulated after combination treatment of ADO and HCY.The mRNA expressions of DNMT1 ,DNMT3 a and DNMT3 b were down-regulated after combination treatment with ADO and HCY or 5-Aza-CdR alone.Conclusion Combination treatment of ADO and HCY can cause cellular methylation chan-ges.The effects of demethylation of ADO and HCY may activate p53 gene and mitochondrial pathway, which at last leads to HepG2 cell apoptosis.