表观遗传学干预对结直肠癌细胞株p33^(ING1b)基因表达及生物学特性影响

Influence of epigenetic intervention on p33^(ING1b) expression and biological characteristics of colorectal cancer cell lines

目的通过表观遗传学干预,研究p33ING1b启动子甲基化和组蛋白乙酰化状态、基因和蛋白的表达及其细胞增殖能力的改变,探讨结直肠癌中p33ING1b转录抑制的表观遗传机制。方法通过曲古抑菌素A及5-氮-2′-脱氧胞苷,对结直肠癌细胞株HT29、LOVO、HCT116和COLO205行恢复乙酰化和去甲基化干预,分单独用药组和联合用药组。RT-PCR结合Real-time qPCR方法检测其p33ING1bmRNA表达的变化,nMSP法分析其p33ING1b启动子甲基化状态的改变,ChIP法检测其p33ING1b乙酰化状态的改变,蛋白质印迹法检测p33ING1b蛋白的表达,MTT法检测细胞的增殖能力。结果曲古抑菌素A及5-氮-2′-脱氧胞苷可逆转结直肠癌细胞株的甲基化及乙酰化状况。2种药物干预后,4种结直肠癌细胞株各组间p33ING1bmRNA表达,差异有统计学意义,HCT116:F=1 690.446,P<0.001;HT29:F=284.474,P<0.001;LOVO:F=2 930.284,P<0.001;COLO205:F=33.540,P<0.001;p33ING1bmRNA蛋白表达差异有统计学意义,HCT116:F=32.606,P<0.001;HT29:F=33.422,P<0.001;LOVO:F=13.975,P<0.01;COLO205:F=7.119,P<0.05,细胞增殖能力差异有统计学意义,HCT116:F=7.327,P<0.05;HT29:F=17.642,P<0.001;LOVO:F=7.035,P<0.05;COLO205:F=9.008,P<0.01。联合使用两种药物对p33ING1bmRNA的表达具有协同作用。结论 p33ING1b基因甲基化和去乙酰化可抑制抑癌基因p33ING1b转录,使用甲基化抑制剂及去乙酰化抑制剂可逆转这一效应,且两者具有协同作用,这为表观基因治疗提供了新的思路。

OBJECTIVE To investigate the epigenetic mechanism of the p33iN1b transcription inhibition through epi- genetic intervention and analyze the change of epigenetic pattern, genetic expression in p33INGlb promoter,protein expres- sion of p33ING1b and proliferation of colorectal cancer cells. METHODS Colorectal cancer cell lines were cultured in vitro and divided into four groups：control group treated without any drug, and the other three experimental groups treated with TSA（TSA group）, 5-Aza-2＇-dc （Aza group）, 5-Aza-2＇-dc ＋ TSA（Aza＋ TSA group） separately. The expressions of p33IN6~b mRNA were detected by real-time quantitative RT-PCR. The patterns of p33~N^IB promoter methylation were ana- lyzed by nMSP. Acetylation levels of p33INGIB fragment 1 and 2 were estimated by CHIP. p33ING1b protein expression was detected by Western Blot. The biological behaviors of the colorectal cancer cells were investigated by MTT. RESULTS TSA and 5-Aza-2Cdc can rerevse methglation and acetylation status of the colorectal cancer lines. After the intervention of TSA and 5-Aza-2Cdc,the differences of p33ING1bmRNA expression in four cell lines were significant（HCTl16：F= 1 690. 446, P〈 0. 001 ; HT29 ： F= 284. 474,P〈0. 001 ; LOVO： F= 2 930. 284,P〈0. 001 ; COLO205 ： F= 33. 540, P〈0. 001） ; The differences of p33tNc^b mRNA protein expression were significant （ HCT116 ： F= 32. 606, P〈 0. 001 ; HT29 ： F = 33. 422, P〈0. 001 ; LOVO： F = 13.975, P〈0. 01 ; COLO205 ： F= 7. 119, P〈 0.05 ）. The proliferation of colorectal cancer ceils were significant （ HCT116 ： F = 7. 327, P〈0. 05 ; HT29 ： F = 17. 642, P〈 0. 001 ; LOVO： F = 7. 035, P〈 0. 05 ; COLO205 ： F = 9. 008, P〈0. 01 ）. There was a synergistic effect with the combination of the two drugs. CONCLUSIONS Methylation and deacetylation inhibit the transcription of p33INGlb , 5-Aza-2＇-dc and TSA can reverse the efficacy and exhibite obvious synergistic effect. It will provide a novel approach to epigenetic therapy.