NS-398调节HepG2细胞组蛋白乙酰化和p21WAF1/CIP1表达

Regulation of Histone Acetylation and the Expression of p21WAF1/CIP1 by NS-398 in HepG2 Cells

背景与目的:研究非甾体药物NS-398对人肝癌细胞株HepG2细胞组蛋白H3乙酰化水平的调节作用及对细胞周期素依赖性激酶抑制p21WAF1/CIP1表达的影响。材料与方法:用不同浓度(100、200、300、400μmol/L)的NS-398处理HepG2细胞,以四甲基偶氮唑蓝(MTT)法测定肿瘤细胞增殖抑制率,流式细胞仪(FCM)检测细胞周期的改变及凋亡百分率的变化,应用NS-398分别作用HepG2细胞4、8、12、24、48h,非药物作用组作为对照,提取细胞的总RNA和总蛋白,采用RT-PCR技术检测p21WAF1/CIP1mRNA表达情况,并用免疫印迹技术(Western blot)观察组蛋白H3的乙酰化水平变化及p21WAF1/CIP1蛋白的表达水平。结果:NS-398抑制HepG2细胞增殖,且呈剂量依赖性,并诱导其凋亡。且呈浓度依赖性改变细胞周期的分布,一方面增高G0/G1期细胞比例,另一方面降低S期和G2/M期细胞比例,与对照组相比差异具有统计学意义(P<0.05)。NS-398对组蛋白H3乙酰化作用随时间改变而变化,可引起组蛋白H3的乙酰化。NS-398对p21WAF1/CIP1 mRNA和p21WAF1/CIP1蛋白表达的影响呈时间依赖性。结论:NS-398明显上调HepG2细胞组蛋白H3的乙酰化水平,促进细胞周期依赖性激酶抑制剂p21WAF1/CIP1的表达。

BACKGROUND ＆ AIM： To investigate the regulation of histone H3 acetylation and the expression of p21WAF1/ CIP1 by NS-398 in HepG2 cells. MATERIALS AND METHODS： The effect of NS-398 on the proliferation of HepG2 cells was evaluated by MTF. The apoptotie cells were determined by flow eytometrie（FCM） analysis.Total proteins and mRNA were extracted from HepG2 cells treated with or without NS-398 with IC50 200 μmol/ L at various time points （4,8,12,24,48 h）,By using Westem blot, the levels of aeetylated histone H3 and p21WAF1/CIP1 protein expression were assayed, RT-PCR was used to detect the expression level of p21WAF1/CIP1 mRNA. RKSULTS： NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with increasing NS-398 concentration. Histone H3 aeetylation was obviously increased by NS-398 in a time-dependent manner. The expression levels of p21WAF1/CIP1 mRNA and protein were up-regulated in a time-and dose-dependent manner. CONCLUSION： The level of aeetylated histone H3 was up-regulated by NS-398, and the expression of p21WAF1/CIP1 was increased at the same time.