摘要目的 观察分泌型卷曲相关蛋白1(SFRP1)基因在非小细胞肺癌(NSCLC)组织中的甲基化状态,研究抑甲基化制剂5-氮杂-2’-脱氧胞苷(5-Aza-CdR)对肺癌SPC-A1细胞中SFRP1、p16和O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因甲基化的影响.方法 采用甲基化特异性PCR(MSP)法和免疫组化SP法,检测60例NSCLC组织中SFRP1基因甲基化的状态和蛋白的表达,以21例肺良性病变组织作为对照.以5-Aza-CdR处理肺癌SPC-A1细胞,采用MSP法和RT-PCR法检测处理前后细胞中 SFRP1、p16和MGMT基因的甲基化状态及相应mRNA的表达.结果 SFRP1基因在60例NSCLC组织中的甲基化率为58.3％,明显高于肺良性病变组织(14.3％;x2 =12.118,P=0.001);SFRP1基因的甲基化与NSCLC的分化程度和淋巴结转移情况有关(均P＜0.05).SFRP1蛋白在35例SFRP1基因甲基化的NSCLC组织中的阴性表达率为68.6％,明显高于非甲基化的NSCLC组织(24.0％；x2=9.613,P=0.002)；SFRP1蛋白的阴性表达与NSCLC的分化程度、临床分期以及淋巴结转移情况有关(均P＜0.05).未用5-Aza-CdR处理时,SPC-A1细胞中SFRP1、p16和MGMT基因甲基化和mRNA的表达量很低；应用不同浓度的5-Aza-dCR处理后,SPC-A1细胞中SFRP1、p16和MGMT基因甲基化和mRNA的表达均显著上调(均P＜0.05).结论 SFRP1基因的甲基化与NSCLC的发生发展密切相关；5-Aza-CdR能逆转SFRP1、p16和MGMT基因的甲基化状态,促进其重新表达.

abstractsObjective To observe the expression of SFRP1 gene methylation in non-small cell lung cancer (NSCLC),and study the effect of 5-Aza-2-deoxycytidine (5-Aza-CdR) on DNA methylation and expression of SFRP1,p16 and MGMT genes in the human lung cancer cell line SPC-A-1 cells.Methods SP immunohistochemistry and methylation-specific PCR were used to detect the SFRP1 methylation in 60 NSCLC cases,and 21 cases of benign lung diseases were used as control group.SPC-A-1 cells were cultured and treated with 5-Aza-CdR.The promoter methylation status of SFRP1,p16 and MGMT genes were detected by methylation-specific polymerase (MSP) chain reaction,and mRNAs were detected by real-time PCR.Results The positive rate of SFRP1 gene methylation in NSCLC was significantly higher than that in normal lung tissue (58.3％ vs.14.3％ ; x2 =12.118,P =0.001).SFRP1 gene methylation was closely correlated with lymph node metastasis and degree of differentiation in NSCLC (P ＜ 0.05).SFRP1 protein expression was correlated with clinical stage,degree of differentiation and lymph node metastasis in NSCLC (P ＜0.05).The positive expression of SFRP1 protein in 30 cases of NSCLC tissue containing SFRP1 gene methylation was significantly higher than that in non-methylated NSCLC (68.6％ vs.24.0％ ; x2 =9.613,P =0.002).SFRP1 gene methylation was closely correlated with SFRP1 gene protein expression in NSCLC (P ＜0.05).Negative expression of SFRP1 protein was correlated with the differentiation,clinical stage,and lymph node metastasis in NSCLC (all P ＜ 0.05).Without 5-Aza-CdR treatment,the expressions of methylation of SFRP1,p16 and MGMT genes and their mRNA were low.After 5-Aza-CdR treatment at different concentrations,their expressions were significantly elevated (all P ＜ 0.05).Conclusions SFRP1 gene methylation is closely associated with carcinogenesis and development of NSCLC.5-Aza-CdR may reverse the methylation of SFRP1,p16 and MGMT genes,and facilitate the re-expression of the anti-oncogenes.