组蛋白脱乙酰基酶抑制剂丁酸钠对胃癌细胞系生长及p16基因表达的影响

Effects of sodium butyrate on proliferation of human gastric cancer cells and expression of p16 gene

目的探讨组蛋白脱乙酰基酶抑制剂丁酸钠（NaB）对体外培养的胃癌细胞系SGC7901和BGC823生长及p16基因表达的影响。方法应用四甲基偶氮唑盐比色法（MTT）检测细胞增殖活性，流式细胞仪检测细胞周期分布，膜联蛋白V-异硫氰酸荧光素染色法检测细胞凋亡率，逆转录聚合酶链反应（RT-PCR）及蛋白质印迹法检测p16基因mRNA及蛋白质表达，甲基化特异性PCR法（MSP）检测p16基因的甲基化状态。结果NaB处理后SGC7901和BGC823细胞生长受到抑制，1×10^-3，3×10^-3，5×10^-3mol／LNaB处理72h后两株细胞G0／G1期细胞数量显著增加，S期细胞数显著降低（P〈0．01），呈现G1阻滞，各浓度组细胞凋亡率均显著增加（P〈0．01）。p16基因在SGC7901及BGC823细胞中低表达，启动子区呈异常甲基化状态，NaB处理后在两株细胞中表达均增强。结论NaB对胃癌细胞具有生长抑制作用，其机制可能与阻滞细胞周期、诱导凋亡及上调p16基因表达有关，NaB可能通过增加组蛋白乙酰化水平及甲基化下调增加胃癌细胞中p16基因的表达。

Objective To investigate the effects of sodium butyrate （NaB）, a histone deacetylase inhibitor （HDACI）, on the proliferation of human gastric cancer cells and the expression of p16 , an important negative-regulatory factor of the cell cycle G1. Methods Human gastric cancer cells of the lines SGC7901 and BGC823 were cultured in RPMI1640 medium and treated with NaB of different concentrations ： 5 × 10^-4 1 × 10^-3, 3 × 10^-3, and 5 × 10^-3 mol/L for 24 h or 72 h. MTT assay, flow cytometry, and annexin V-fluorescein isothiocyanate staining were performed to analyze the cell proliferation activity, cell cycle, and apoptotic rate. RT- PCR, Western blotting, and methylation-specific PCR （MSP） were used to detect the e mRNA and protein expression and methylation state of p16 gene respectively. Results Exposure to different concentrations of NaB inhibited the growth of the SGC7901 and BGC823 cells. Treated with NaB of the concentrations of 1 × 10^-3 , 3 × 10^-3 , and 5 × 10^-3 mol/L respectively for 72 h, the numbers of the SGC7901 and BGC823 cells at G0/G1 stage increased significantly and those at S stage decreased significantly （ all P 〈 0.01 ）, which showed that the cell cycle was blocked at G1 phase. The apoptosis rates of the SGC7901 and BGC823 cells treated with NaB of different concentrations for 72 h all increased significantly （all P 〈0.01 ）. P16 was expressed in the SGC7901 and BGC823 cells at low levels, and hypermethylation was detected in its promoter region in both cells before the treatment of NaB. After treated with NaB the mRNA and protein expressions of p16 gene were increased in both cells. Conclusion NaB inhibits the growth of human gastric cancer cells by blocking cell cycles, inducing apoptosis and upregulating the expression of p16 gene by increasing acetylation and reducing methylation.