应用丙戊酸钠调节组蛋白乙酰化修饰抑制肝癌细胞增殖的作用及机制

Effect and mechanism of regulating histone acetylizad level VPA on the proliferation of hepatocellular carcinoma cells

目的观察应用组蛋白脱乙酰酶抑制剂丙戊酸钠调节染色体组蛋白低乙酰化水平对肝癌细胞增殖的作用，并进一步检测Cyctin A、Cyclin D1、Cyclin E、P21^Waf/cip1蛋白及mRNA表达变化，探讨其分子作用机制。方法应用0．75～4．0mmol／L丙戊酸钠作用于肝癌细胞系HepG2细胞后，MTT法检测细胞生长抑制、细胞克隆形成试验观察克隆形成率、碘化丙啶标记流式细胞术检测细胞周期、间接免疫荧光法分析Cyclin A、Cyclin D1、Cyclin E、P21^Waf/cip1蛋白表达，RT—PCR检测分析Cyclin A、Cyclin D1、Cyclin E、P21^Waf/cip1 mRNA表达。结果0．75～4．0mmol／L丙戊酸钠作用24、48、72、96h，观察组出现了时间-剂量依赖趋势的生长抑制，同时细胞克隆形成率显著降低，对照组细胞增殖周期G1、S、M期所占比例未见明显改变，而观察组则随药物浓度、作用时间的不同而出现不同程度的细胞增殖周期G1期阻滞，G1期比例由55．4％～82．8％不等，差异有统计学意义（P〈0．01）。观察组Cyclin A、Cyclin D1蛋白及mRNA表达均被明显下调而P21^Waf/cip1蛋白、mRNA表达则被明显上调，与对照组比较差异均有统计学意义（均P〈0．01）；而Cyclin E蛋白和mRNA表达变化则未见明显差异（P〉0．05）。结论通过应用特异性组蛋白脱乙酰酶抑制剂调节组蛋白乙酰化修饰可明显抑制肝癌细胞生长、抑制细胞克隆形成、阻滞细胞增殖周期于G1期；丙戊酸钠作为组蛋白脱乙酰酶抑制剂可明显抑制肝癌细胞增殖，其作用机制是通过上调P21^Waf/cip1 mRNA蛋白表达，下调Cyclin D1、Cyclin A mRNA蛋白表达分别和／或协同实现。

Objective To investigate the suppressing effect on hepatocellular carcinoma cell proliferation by up-regulating histone acetylizad level with a selective inhibitor of HDACs-Valproate acid sodium（VPA）. And the mechanism which involved was clarified by detecting the protein and mRNA expressions of Cyclin A,Cyclin D1 ,Cyclin E,P21^Walf/cip1. Methods HepG2 cells were cultured with 0.75-4.0 mmol/L valproic acid （VPA） for 24,48, 72,96hrs in vitro. The inhibition rate was studied by MTT assay; clone forming inhibit rate was tested by clonay assay; cell cycle was analyzed by flow cytometry with PI assay, and the protein and mRNA expressions of Cyclin A, Cyclin D1 ,Cyclin E,P21^Waf/cip1 of HepG2 cells after 1.5,3.0 mmol/L VPA treated were analyzed by indirect immunofluorescence technique and RT-PCR respectively. Results The inhibition rate of experimental group increased significantly（ P 〈 0. 001 ） and a dose and acting time dependence was found. Clones in experimental groups decreased more than in control group. As for cell cycle, the percentage of G1, S, M phrase in control group remained the same. Compared with control group, 0.75 and 4.0 mmol/L VPA induced a significant arrest in G1 phrase（P 〈 0. 001） and a total of 55.4% - 82.8% G1 phrase of cells which cultured with VPA. Cyclin A, Cyclin D1 were down-regulated both at mRNA and protein level （P 〈 0. 001 ）while P21^Waf/cip1 was up-regulated both at mRNA and protein level. Conversely, neither mRNA nor protein expression of Cyclin E showed any difference between experimental and control group （P 〉 0.05）. Conclusion Up-regulating histone acetylizad level can inhibit hepatocellular carcinoma cell proliferation, inhibit cell clone formation, induce cell cycle arrest in G1 phrase. VPA, as a I class of histone deacetylase inhibitor can be used as an option in the treatment of hepetoma. The mechanism includes upregulating P21^Waf/cip1 mRNA and protein expression, down-regulating Cyclin A , Cyclin D1 mRNA and protein expression.