GPX3基因甲基化是胰腺癌的潜在诊断标志物

Methylation of GPX3 in promoter region is a potential pancreatic cancer diagnostic marker

目的 探讨谷胱甘肽过氧化物酶3(glutathione peroxidase 3,GPX3)基因启动子区在胰腺癌中的甲基化情况及其表达调控机制。方法 应用半定量RT-PCR、甲基化特异性PCR(methylation specific PCR,MPCR)和硫化测序(bisulfite sequencing,BSSQ)技术对6株人胰腺癌细胞系(PANC-1、PANC3.11、MIAPaCa-2、SW1990、AsPC-1和CFPAC-1)和153例胰腺癌组织进行分析。结果GPX3在PANC-1、PANC3.11和SW1990细胞中高表达,其启动子区呈现非甲基化状态;在CFPAC-1细胞中低表达,其启动子区呈现部分甲基化状态;在MIAPaCa-2和AsPC-1细胞中缺失表达,其启动子区呈现完全甲基化。应用去甲基化试剂5-aza处理后,PANC-1、PANC3.11和SW1990细胞中表达无变化,CFPAC-1细胞中表达增加,MIAPaCa-2和AsPC-1细胞中恢复表达。结果表明GPX3的表达受启动子区甲基化调控。BSSQ结果进一步验证了其在细胞系中的状态和MSP的扩增效率。GPX3在胰腺癌中的甲基化率为26.1%(40/153),GPX3甲基化与年龄、吸烟和肿瘤分化程度有相关性(P<0.05),与性别、肿瘤大小、TNM分期、饮酒等因素无相关性(P>0.05),GPX3甲基化是胰腺癌诊断的潜在标志物。结论 GPX3在胰腺癌中频繁发生甲基化,其表达受启动子区甲基化调控。

Objective To investigate the methylation status of glutathione peroxidase 3(GPX3) and the regulation of expression in pancreatic cancer.Methods 6 pancreatic cancer cell lines(PANC-1,PANC3.11,MIAPaCa-2,SW1990,AsPC-1 and CFPAC-1) and 153 cases of pancreatic cancer samples were employed.Semi-quantitative RT-PCR,methylation specific PCR(MSP) and bisulfite sequencing(BSSQ) techniques were applied to detect the expression and methylation status of GPX3.Results GPX3 was high expressed in PANC-1,PANC3.11 and SW1990 cells,and the promoter region was unmethylated;GPX3 was reduced expressed in CFPAC-1 cells,the promoter region was partial methylated;GPX3 was unexpressed in MIAPaCa-2 and AsPC-1 cells,the promoter region was completely methylated.The expression of GPX3 was restored in MIAPaCa-2 and AsPC-1 cells,increased expression was found in CFPAC-1 cells,and no expression changes were found in PANC-1,PANC3.11 and SW1990 cells after treatment with 5-aza-2′-deoxycytidine(5-aza).The methylation status was validated by BSSQ in MIAPaCa-2,AsPC-1,PANC-1 and SW1990 cells.The results suggested that the expression of GPX3 was regulated by promoter region methylation in pancreatic cells.GPX3 was methylated in 26.1%(40/153) of pancreatic cancer.Methylation of GPX3 was associated with age,smoking and cancer differentiation(all P<0.05),no association with gender,tumor size,drinking and TNM stage(all P>0.05).Methylation of GPX3 may serve as a potential diagnostic marker of pancreatic cancer.Conclusion GPX3 is frequently methylation in pancreatic cancer,and its expression is regulated by promoter region methylation.