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目的 :探讨Ras相关区域家族5(Ras-association domain family 5,RASSF5)基因不同转录本在食管鳞癌(esophageal squamous cell carcinoma,ESCC)中的表达及其甲基化状态。方法 :应用RT-PCR法检测49例食管鳞癌组织及其癌旁正常组织中RASSF5基因不同转录本mRNA的表达及DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycitydine,5-aza-dC)处理前后食管癌细胞TE1、TE13、T.Tn和Ec109中RASSF5A mRNA的表达;应用甲基化特异性PCR(methylation specii c polymerase chain reaction,MSP)法检测49例食管鳞癌组织及其癌旁正常组织和5-aza-dC处理前后食管癌细胞中RASSF5A甲基化状态,应用免疫组织化学法检测49例食管鳞癌组织及其癌旁正常组织中RASSF5A蛋白的表达。结果 :RASSF5A转录本是在食管鳞状上皮中表达的主要转录本。经5-aza-dC处理后,TE1、TE13、T.Tn和Ec109细胞中RASSF5A mRNA的表达水平均上调(P<0.05);TE1细胞中RASSF5A基因甲基化程度降低,非甲基化程度增加,TE13、T.Tn和Ec109细胞中RASSF5A基因均表现为非甲基化状态。食管鳞癌组织中RASSF5A mRNA的表达水平明显低于癌旁正常组织(P<0.01),其蛋白的阳性表达率(26.5%,13/49)也显著低于癌旁正常组织(75.5%,37/49)(P<0.01)。食管鳞癌组织中RASSF5A基因启动子区的甲基化率(55.1%,27/49)显著高于癌旁正常组织(28.6%,14/49)(P<0.05),并与淋巴结转移和肿瘤组织的分化程度有关(P<0.01)。结论 :食管鳞状上皮组织中RASSF5的主要转录本可能是RASSF5A,RASSF5A基因启动子区异常高甲基化导致的基因沉默可能与食管鳞癌的发生、转移及恶性进程有关。
Objective: To investigate the expression and methylation status of different transcripts of Ras-association domain family 5 (RASSF5) gene in esophageal squamous cell carcinoma (ESCC). Methods: The expressions of RASSF5 mRNA in 49 esophageal squamous cell carcinomas and adjacent normal tissues were detected by RT-PCR, and the expressions of 5-aza-2'-deoxycytidine (5-aza-2'-deoxycytidine) The expression of RASSF5A mRNA in esophageal cancer cells TE1, TE13, T.Tn and Ec109 was detected by Western blotting, and the methylation-specific PCR (MSP) was used to detect the expression of RASSF5A mRNA in esophageal cancer cells before and after treatment with 5-aza-2a-deaza- ) Method was used to detect the methylation status of RASSF5A in 49 cases of esophageal squamous cell carcinoma and its adjacent normal tissues and 5-aza-dC before and after esophageal cancer cells. Immunohistochemistry was used to detect the expression of RASSF5A in 49 esophageal squamous cell carcinoma tissues and normal tissues RASSF5A protein expression in tissues. Results: RASSF5A transcripts were the major transcripts expressed in esophageal squamous epithelium. The expression of RASSF5A mRNA in TE1, TE13, T.Tn and Ec109 cells was up-regulated by 5-aza-dC treatment (P <0.05). The methylation of RASSF5A gene was decreased and the degree of non-methylation was increased in TE1 cells , RASSF5A gene in TE13, T.Tn and Ec109 cells all showed unmethylated status. The positive expression rate of RASSF5A mRNA in esophageal squamous cell carcinoma was significantly lower than that in adjacent normal tissues (26.5%, 13/49) /49)(P<0.01). The methylation rate of RASSF5A gene promoter region (55.1%, 27/49) in esophageal squamous cell carcinoma was significantly higher than that in adjacent normal tissues (28.6%, 14/49) (P <0.05), and was associated with lymph node metastasis and tumor Tissue differentiation degree (P <0.01). CONCLUSIONS: The major transcript of RASSF5 in esophageal squamous epithelium may be the gene silencing induced by aberrant hypermethylation of RASSF5A and RASSF5A gene promoter may be related to the occurrence, metastasis and malignant process of esophageal squamous cell carcinoma.