丙戊酸钠对Molt-4细胞增殖的影响及其作用途径探讨

Investigation on the mechanisms of p15INK4B gene demethylation by valproate in Molt-4 cells

目的 观察丙戊酸钠（VPA）对人急性T细胞白血病细胞系Molt-4细胞增殖和细胞周期的影响,并探讨其对Molt-4细胞p15INK4B基因甲基化状态的影响.方法 不同浓度VPA作用Molt-4细胞后,用MTr法检测细胞增殖抑制率,流式细胞术检测细胞周期的变化,半巢式甲基化特异PCR（MSP）法检测p15INK4B基因甲基化状态,RT-PCR方法检测p15基因、DNA甲基转移酶DNMT-1、DNMT3A、DNMrT3B mRNA的表达.结果 VPA可明显抑制细胞增殖,影响Molt-4细胞周期分布,5.0mmol/L VPA作用Molt-4细胞48 h,G0/G1期细胞为（66.87±3.31）%,S期细胞为（8.47±2.56）%,与未加VPA组相比,S期细胞明显下降,G0/G1期细胞明显升高（P＜0.05）.经VPA作用后Molt-4细胞p15基因甲基化程度明显下降,p15基因表达水平明显增加,DNMT-1、DNMT3B mRNA表达水平下降,以DNMT-1下降较明显.结论 VPA可能通过抑制甲基转移酶DNMT-1和（或）DNMT3B表达使p15INK4B基因去甲基化,使p15基因表达上调,从而抑制Molt-4细胞增殖,使细胞周期阻滞于G0/G1期.

Objective To study the antitumour effects of sodium valproate（VPA） on the proliferation, differentiation and cell cycle of Molt-4 cell and to investigate its demethylation mechanisms. Methods After Molt-4 cells trated with VPA at different concentrations, cell viability and growth curve were assessed by MTT assay. Cell cycle changes were analyzed by flow cytometry. The expression level of p15, DNA methyltransferase 1 （DNMT-1）, DNMT3A and 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR. Results VPA significantly inhibited the proliferation of Molt-4 cells. After 48 h culture with 5.0 mmol/L VPA, the percentages of Molt-4 cells in G0/G1 phase was （66.87 ± 3.31 ） % and in S phase was （8.47 ±2.56）%, while in control group, the cells in G0/G1 phase increased and in S phase decreased significantly. The p15 gene in Molt-4 cells failed to express due to its hypermethylation. The expression level of p15 gene mRNA increased significantly after exposure to VPA for 48 h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner. The expression level of DNMT3B decreased at 10.0 mmol/L concentration. Conclusion VPA has a demethylation effect on p15INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities to recover p15 gene activity, which arrests Molt-4 cell in G0/G1 phase.