应用丙戊酸钠调节组蛋白乙酰化对肿瘤细胞周期相关蛋白的调控作用

Acetylization of hlstone regulated by valproic acid sodium on the regulatory effect of cell cycle related factor Sill

目的探讨应用组蛋白脱乙酰酶抑制剂丙戊酸钠（VPA）调节染色体组蛋白低乙酰化修饰水平对肿瘤细胞增殖周期相关蛋白CyclinA、CyclinD1、CyclinE和P21^Waf/cip1。的调控作用。方法应用0．75—4．00mmol／LVPA干预肝癌细胞HepG2、胃癌细胞BGC-823、乳腺癌细胞MCF-748h后，PI标记流式细胞术检测细胞周期；间接免疫荧光法分析CyclinA、CyclinD1、CyclinE、P21^Waf/cip1。蛋白表达；RT—PCR检测分析CyclinA、CyclinD1、CyclinE、P21^Waf/cip1 mRNA表达。结果HepG2、BGC-823、MCF-7这3种细胞系培养48h后，流式细胞术分析可见0．75～4．00mmol／LVPA实验组随药物浓度的增加而出现逐渐递增的细胞增殖周期G1期阻滞趋势。HepG2、BGC-823细胞CyclinA蛋白及mRNA表达被明显下调；MCF-7细胞CyclinA蛋白及mRNA表达在两个浓度组均未见明显变化；CyclinD1蛋白及mRNA表达在3个细胞系均被明显下调；P21^Waf/cip1蛋白及mRNA表达在3个细胞均被明显上调；CyclinE蛋白及mRNA表达则未见明显变化。结论应用VPA干预组蛋白乙酰化修饰可对HepG2、BGC-823、MCF-7细胞Cyc—linD1、P21^Waf/cip1表达起明显的调控作用；对CyclinA的调控作用则随肿瘤细胞来源及表型的不同而有所差异，而对CyelinE则无明显的调控作用。在VPA诱导肿瘤细胞增殖周期G，期阻滞过程中，下调CyclinD1和上调P21^Waf/cip1蛋白及mRNA表达可能是其共同作用途径。

Objective To investigate the regulation on cell cycle related factor such as Cyclins and P21^Waf/cip1 by inhibiting histone deacetylase （HDAC） with valproic acid sodium（VPA）. Methods HepG2 hep- tocellular carcinoma cells, BGC-823 gastric carcinoma cells and MCF-7 breast cancer cells were cultured with 0.75-4.00 mmol/L VPA for 48 h in vivo, and the cell cycle was analyzed by flow cytometry with PI assay. The protein and mRNA expression of Cyclin A, Cyclin D1, Cyclin E and P21^Waf/cip1 were analyzed by indirect immu- nofluorescence technique and RT-PCR, respectively. Results Compared with control groups, VPA at concentrations 0.75-4.00 mmol/L exerted a significant inhibiting effect on G1 phase of HepG2, BGC-823 and MCF-7 cells（P 〈0.001 ）, and the effect was dose dependent. Cyclin A was down-regulated both at mRNA and protein level in HepG2 and BGC-823 cells（ P 〈 0.001 ）, but no difference in MCF-7 cells（ P 〉 0.05 ）. Cyclin D1 was down-regulated both at mRNA and protein level（ P 〈 0.001 ） and P21^Waf/cip1 was up-regulated both at the mRNA and protein level in the three cell lines（P 〈0. 001 ） ; Conversely, protein and mRNA expression of Cyclin E were unchanged upon treatment with VPA （P 〉 0.05 ）. Conclusion Acetylization of histone intervened with VPA can regulate Cyclin D1 and P21^Waf/cip1 expressions obviously. To the expression of Cyclin A, it shows some difference according to the histogenesis and phenotypes of different carcinoma types. But there is not any obvious function on Cyclin E. Down-regulating Cyclin D1 and up-regulating P21^Waf/cip1 may be the common target pathway in the inhibition of cell cycle G1 phase exerted by VPA.