目的:探讨Ras相关区域家族5(Ras-associationdomainfamily5,融SSF5)基因不同转录本在食管鳞癌(esophagealsquamouscellcarcinoma,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融SSF5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RASSF5AmRNA的表达;应用甲基化特异性PCR(methylation specific 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见4.S.SF54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Ecl09细胞中RASSF5AmRNA的表达水平均上调(P〈0.05);TE1细胞中尼4SSF5月基因甲基化程度降低,非甲基化程度增加,TEl3、TTn和Ecl09细胞中RASSFSA基因均表现为非甲基化状态。食管鳞癌组织中RASSF5AmRNA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〈O.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,RASSF54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 expressions and methylation status of different transcripts of Ras-association domain family 5 (RASSF5) in esophageal squamous cell carcinoma (ESCC). Methods: The expressions of different transcript mRNAs of RASSF5 in 49 ESCC tissues and the corresponding paracancerous normal tissues and the expression of RASSF5A mRNA in esophageal cancer TE1, TE13, T.Tn and Ec109 cells treated or untreated with 5-aza-2'-deoxycitydine (5-aza-dC) were detected by RT-PCR. The methylation status of RASSF5A in 49 ESCC tissues and the corresponding paracancerous normal tissues and the esophageal cancer cells treated or untreated with 5-aza-dC were detected by methylation-specific polymerase chain reaction (MSP) method. The expression of RASSF5A protein in 49 ESCC tissues and the corresponding paracancerous normal tissues was examined by immunohistochemistry method. Results: RASSF5A was the main transcript that expressed in esophageal squamous epithelium. The expression levels of RASSF5A in TE1, TE13, T.Tn and Ec109 cells after treatment with 5-aza-dC were up-regulated (P 〈 0.05). The methylation level of RASSF5A was decreased and the unmethylation level of RASSF5A was increased in TEl cell, while the unmethylation status of RASSF5A gene was observed in TEl3, T.Tn and Ec109 cells. The expression level of RASSF5A mRNA in ESCC tissues was lower than that in the corresponding paracancerous normal tissues (P 〈 0.01). The positive expression rate of RASSF5A protein in ESCC tissues (26.5%, 13/49) was significantly lower than that in the corresponding paracancerous normal tissues (75.5%, 37/49) (P 〈 0.01). The methylation rate of RASSF5A promoter in ESCC tissues (55.1%, 27/49) was significantly higher than that in the corresponding paracancerous normal tissues (28.6%, 14/49) (P 〈 0.05), and the methylation rate was closely correlated with lymph node metastasis and pathological differentiation (P 〈 0.01). Conclusion= The main transcript of RASSF