IGFBP3基因在食管鳞状细胞癌中的表达及甲基化状态

Expression and methylation of IGFBP3 gene in esophageal squamous cell cancer

目的:检测食管鳞状细胞癌(esophageal squamous cell cancer,ESCC)中胰岛素样生长因子结合蛋白3(insulin-like growth factor binding protein 3,IGFBP3)基因的表达情况及甲基化状态,探讨其与ESCC发生发展的关系。方法:收集河北医科大学第四医院2008至2011年间的82例ESCC手术患者的ESCC原发灶组织及癌旁正常黏膜组织。RT-PCR及甲基化特异性-PCR(methylation specific-PCR,MSP)的方法分别检测DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycitydine,5-Aza-d C)处理前后的ESCC细胞系(TE1、TE13、YES-2、T.TN、Eca109)及82例ESCC及相应癌旁组织中IGFBP3基因mRNA表达水平及甲基化状态,应用免疫组织化学方法检测IGFBP3在ESCC组织中的蛋白表达情况,并分析IGFBP3基因甲基化状态与其表达水平之间的关系。结果:在ESCC细胞株TE1、TE13、YES-2、T.TN、Eca109中,IGFBP3基因mRNA均呈阴性或弱阳性表达,用5-Aza-d C培养处理后,其mRNA表达水平均呈现不同程度的增高(P<0.05);MSP检测结果显示,在ESCC细胞株TE1、TE13、T.Tn、Yes-2中IGFBP3基因均呈高甲基化状态。在ESCC组织中IGFBP3 mRNA表达显著低于癌旁组织[(0.15±0.07)vs(0.88±0.32),P<0.01],且IGFBP3蛋白在癌组织中的表达阳性率显著低于癌旁组织[29.3%(24/82)vs84.1%(69/82),P<0.01],并与TNM分期密切相关(P<0.05);IGFBP3基因在ESCC组织中的甲基率为68.3%(56/82),明显高于癌旁组织的15.9%(13/82)(P<0.01);IGFBP3基因在Ⅲ和Ⅳ期肿瘤组织中的甲基化率明显高于Ⅰ和Ⅱ期肿瘤组织(P<0.05),而该基因的甲基化率与肿瘤患者的组织学分级无相关性(P>0.05)。IGFBP3基因甲基化状态与其表达之间有明显的相关性(P<0.05)。结论:ESCC组织及细胞株中IGFBP3基因呈高甲基化状态,该基因的甲基化可能导致其表达下调,并有可能是ESCC的发生机制之一。

Objective: To investigate the expression and methylation of IGFBP3 gene in esophageal squamous cell carcinoma( ESCC) cell lines and primary tumor tissues,and to explore the relationship between IGFBP3 expression and the clinical pathological features of ESCC. Methods: The mRNA and methylation status of IGFBP3 gene were detected by reverse transcription-PCR( RT-PCR) and methylation specific-PCR( MSP) using RNA and DNA from ESCC cell lines( TE1,TE13,YES-2,T. TN,Eca109) as well as primary tumor tissues and paired normal tissues from 82 ESCC patients.Immunohistochemistry was used to detect the expression of IGFBP3 in ESCC tissues. The relationship among aberrant methylation,expression of IGFBP3 gene,and clinical pathological features were analyzed with the SPSS 13. 0 software.Results: IGFBP3 mRNA was undetectable or at very low level in ESCC cell lines examined( TE1,TE13,YES-2,T. TN,Eca109). However,its level increased significantly after the cells were treated with DNA methyltransferase inhibitor,5-aza-2'-deoxycytidine( 5-Aza-d C),indicating that IGFBP3 gene existed is hypermethylated in these cells( P < 0. 05). In primary tumor tissues from ESCC patients,IGFBP3 mRNA level( 0. 15 ± 0. 07) was significantly lower than that in corresponding normal tissues( 0. 88 ± 0. 32)( P < 0. 01). Similarly,the positive rate of IGFBP3( 29. 3%,24 /82) in ESCC tissues was significantly lower than that in corresponding normal tissues( 84. 1%,69 /82)( P < 0. 01). Thus,The methylation status of IGFBP3 gene associates with its mRNA and protein expression( P < 0. 05). Moreover,the methylation frequency of IGFBP3 gene in ESCC tissues( 68. 3%,56 /82) was increased significantly compared to that in corresponding normal tissues( 15. 9%,13 /82)( P < 0. 01) and associated with TNM stage of the tumors( P < 0. 05). The methylation frequency of IGFBP3 gene in stage Ⅲ-Ⅳ tumor tissues was significantly higher than that in stageⅠ-Ⅱtumor tissues( P < 0. 05). However,the methylation status of IGFBP3 in ESCC tissues was not associated with its pathological grade( P > 0. 05). Conclusion: The hypermethylation of IGFBP3 gene is a frequent event in ESCC cell lines and primary tumor tissues. The reduced expression of IGFBP3 caused by promoter hypermethylation of the gene may play an important role in the development of ESCC.