醋酸棉酚对人腺样囊性癌ACC-M细胞P16基因甲基化及mRNA表达的影响及其意义

Effects of Gossypol Acetic Acid on DNA Methylation Level and mRNA Expression of p16 Gene in Human Adenoid Cystic Carcinoma ACC-M Cells

目的研究醋酸棉酚(gossypol acetic acid,GAA)对体外培养的人腺样囊性癌ACC-M细胞P16基因甲基化及mRNA表达的影响,初步探讨醋酸棉酚的抗肿瘤作用机制.方法 (1)应用甲基化特异性PCR(MSP)检测ACC-M细胞在GAA作用48、72 h后P16基因甲基化状态的改变情况;(2)应用实时荧光定量法(RFQ-PCR)检测ACC-M细胞在GAA作用48、72 h后P16基因mRNA的表达变化.结果 (1)MSP检测结果显示:分别以19、10μmol/L的GAA作用ACC-M细胞48、72 h后,P16甲基化条带的平均灰度值低于对照组(P<0.05);(2)RFQ-PCR检测显示:分别以19、10μmol/L的GAA作用ACC-M细胞48、72 h后细胞中P16mRNA的表达水平高于对照组(P<0.05).结论 GAA可降低P16基因的甲基化水平,且能增强P16基因mRNA表达,这可能是GAA抗肿瘤作用的机制之一.

Objective To study the effects of gossypol acetic acid （GAA） on methylation level and mRNA expression of p16 gene in human adenoid cystic carcinoma ACC-M cells in vitro,and investigate the antitumor mechanism of GAA.Methods （1） Methylation specific PCR （MS-PCR） was used to detect methylation level changing of the p16 in ACC-M cells treated by GAA for 48 h and 72 h.（2） Real-time fluorescence quantitative PCR （RFQ-PCR） was used to detect mRNA expression level changing of the p16 in ACC-M cells treated by GAA for 48 h and 72 h.Results （1） Compared with the control group,MS-PCR showed the hypermethylation of CpG island in gene promoter ofp16 was partly reversed after GAA treatment （19 μmol/L GAA for 48 h and 10 μmol/L for 72 h,P ＜ 0.05） （2） The results of RFQ-PCR showed that mRNA expression of p16 in ACC-M cells was significantly higher than that in the control group after GAA treatment （19 μ mol/L GAA for 48 h and 10 μmol/L for 72 h,P ＜ 0.05） Conclusion GAA can reduce the p16 gene methylation level and increase p16 gene mRNA expression,which might be one of the mechanisms of GAA antitumor activity.