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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 detect the expression and methylation status of insulin-like growth factor binding protein 3 (IGFBP3) in esophageal squamous cell carcinoma (ESCC) The development of the relationship. Methods: The ESCC primary tumor tissues and adjacent normal mucosa tissues of 82 ESCC patients from 2008 to 2011 in the Fourth Hospital of Hebei Medical University were collected. RT-PCR and methylation-specific PCR (MSP) were used to detect the DNA methyltransferase inhibitor 5-aza-2’-deoxycitydine The expression of IGFBP3 mRNA in ESCC cell lines (TE1, TE13, YES-2, T.TN and Eca109) and 82 ESCC and corresponding paracancerous tissues before and after 5-Aza- Immunohistochemistry was used to detect the protein expression of IGFBP3 in ESCC tissues and to analyze the relationship between methylation status of IGFBP3 gene and its expression level. Results: IGFBP3 mRNA expression was negative or weakly positive in ESCC cell lines TE1, TE13, YES-2, T.TN and Eca109, and the expression of IGFBP3 mRNA was different after 5-Aza-d C culture (P <0.05). The results of MSP showed that IGFBP3 gene was hypermethylated in ESCC cell lines TE1, TE13, T.Tn and Yes-2. The expression of IGFBP3 mRNA in ESCC was significantly lower than that in paracancer tissues [(0.15 ± 0.07) vs (0.88 ± 0.32, P <0.01], and the positive rate of IGFBP3 in cancer tissues was significantly lower than that in paracancerous tissues [29.3 % (24/82) vs84.1% (69/82), P <0.01], and was closely related to TNM stage (P <0.05). The methylation rate of IGFBP3 in ESCC tissues was 68.3% (56/82 ) Was significantly higher than that in paracancerous tissues (15.9%, 13/82) (P <0.01). The methylation rates of IGFBP3 gene in stage Ⅲ and Ⅳ tumors were significantly higher than those in stage Ⅰ and Ⅱ tumors (P <0.05) ), While the methylation rate of this gene had no correlation with the histological grade of tumor patients (P> 0.05). There was a significant correlation between the methylation status of IGFBP3 gene and its expression (P <0.05). CONCLUSIONS: The IGFBP3 gene is highly methylated in ESCC tissues and cell lines. Methylation of this gene may result in down-regulation of IGFBP3 gene expression and may be one of the mechanisms of ESCC.