5-Aza-dC及TSA对人胃癌细胞株SGC-7901 Runx3基因甲基化及表达水平的影响

Effect of 5-Aza-2'-deoxycytidine and trichostatin A on expression and methylation of the Runx3 gene in human gastric carcinoma line SGC-7901

目的:探讨5-Aza-dC及TSA对人胃癌细胞系SGC-7901中抑癌基因Runx3启动子区甲基化、mRNA及蛋白表达水平的影响.方法:单独或联合应用5-Aza-dC及TSA处理体外培养的SGC-7901细胞,提取各组细胞的DNA、RNA及蛋白质,应用甲基化特异性定量PCR法(QMSP)检测Runx3基因启动子区甲基化状态,逆转录PCR法(RT-PCR)检测Runx3mRNA的表达,免疫印迹法(Western blotting)法检测Runx3蛋白表达水平.结果:5-Aza-dC和TSA均能降低Runx3基因启动子区的甲基化水平(5-Aza-dC组及TSA组分别为对照组的0.70倍、0.63倍),提高mRNA表达水平(0.29±0.01、0.28±0.03vs0.14±0.03,P<0.05)及蛋白表达水平(0.35±0.02、0.37±0.02vs0.09±0.01,P<0.05);与单独使用5-Aza-dC和TSA相比,两药联合组Runx3基因启动子区甲基化水平(对照组的0.37倍)及mRNA表达水平(0.45±0.02)和蛋白表达水平(0.50±0.01)均较单药组效果更明显(P<0.05).结论:5-Aza-dC和TSA均能逆转胃癌细胞SGC-7901 Runx3基因的甲基化水平,恢复其mRNA和蛋白表达,且具有协同作用,为5-Aza-dC和TSA应用于胃癌的临床治疗提供了试验依据.

AIM： To evaluate the effect of 5-Aza-2＇-deoxycitydine （5-Aza-dC） and trichostatin A （TSA） on the methylation and expression of the Runx3 gene in human gastric cancer cell line SGC-7901. METHODS： After cultured SGC-7901 cells were treated with 5-Aza-dC and TSA, the methylation levels of the promoter region of the Runx3 gene were detected by quantitative real-time methylation-specific polymerase chain reaction （QMSP）, and Runx3 mRNA and protein expression was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, respectively. RESULTS： Treatment with 5-Aza-dC or TSA alone reduced the methylation levels of the promoter region of the Runx3 gene （70%, 63% vs 100%） and increased Runx3 mRNA （0.29 ± 0.01, 0.28 ± 0.03 vs 0.14 ± 0.03, both P 0.05） and protein expression levels （0.35 ± 0.02, 0.37 ± 0.02 vs 0.09 ± 0.01, P 0.05） compared to control cells. Treatment with 5-Aza-dC in combination with TSA could more signifi cantly reduce Runx3 gene promoter methylation levels （37%） and increase Runx3 mRNA （0.45 ± 0.02） and protein expression levels （0.50 ± 0.01） compared to cells treated with 5-Aza-dC or TSA alone （all P 0.05）. CONCLUSION： 5-Aza-dC and TSA can synergistically reverse Runx3 gene methylation and recover Runx3 mRNA and protein expression in SGC-7901 cells.