口腔鳞状细胞癌RASSF5A基因的甲基化及临床意义

Methylation status and expression of RASSF5A in oral squamous cell carcinoma

目的观察口腔鳞状细胞癌(鳞癌)组织及细胞中Ras相关区域家族5A(RASSF5A)基因的甲基化,并探讨其临床意义。方法收集口腔鳞癌组织及其对应的口腔正常黏膜组织标本49例份;常规培养口腔癌细胞系(OC3、KB),并用5-氮杂-2'-脱氧胞苷(5-Aza-dC)进行处理。采用甲基化特异性PCR技术检测组织与细胞中RASSF5A基因的甲基化状态,用实时荧光定量PCR技术检测组织与细胞中RASSF5AmRNA表达;分析RASSF5A甲基化状态与患者临床病理参数、预后的关系。结果口腔鳞癌组织中RASSF5AmRNA相对表达量低于口腔正常黏膜组织(P<0.001),基因甲基化率高于口腔正常黏膜组织(P=0.003)。5-Aza-dC处理后,OC3、KB细胞中RASSF5AmRNA相对表达量较处理前升高(P<0.05),甲基化程度降低。口腔鳞癌组织中RASSF5A基因甲基化率与肿瘤淋巴结转移、临床分期有关(P均<0.05)。RASSF5A甲基化患者生存率、生存时间均低于未甲基化患者(P均<0.05)。结论口腔鳞状细胞癌组织及细胞中RASSF5A基因启动子区CpG岛高甲基化可能与肿瘤的发生发展有关,且可作为患者预后的评价指标之一。

Objective To detect the expression and methylation status of RASSF5A gene in human oral cancer cell lines and oral squamous cell carcinoma (OSCC) tissues,and to explore the role of RASSF5A gene in occurrence and development of OSCC.Methods Forty-nine OSCC tissues and corresponding normal tissues were taken from patients with OSCC.Oral cancer cell lines (OC3 and KB) were treated by 5-Aza-dC.The methylation status of RASSF5A gene in oral cancer cell lines and OSCC and normal tissues was identified by methylation-specific PCR (MSP).The real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the relative mRNA expression of RASSF5A gene in the oral cancer cell lines and OSCC tissues and the corresponding normal tissues.Meanwhile the correlation between the methylation status and mRNA expression of RASSF5A gene and clinical parameters was analyzed. Results RASSF5A mRNA was significantly lower in the OSCC tissue than that in the corresponding normal tissues ( P <0.001).The frequency of RASSF5A gene methylation was significantly higher in the OSCC tissue than in the normal tissues ( P =0.003).After 5-Aza-dC treatment,the relative expression of RASSF5A in two treated cell lines increased,the methylation status of RASSF5A decreased.The hypermethylation of this gene was related to the lymph node metastasis and clinical stages (all P <0.05).The survival rate and survival time of patients with RASSF5A methylation were lower than those of unmethylated patients (both P <0.05) Conclusion Hypermethylation of CpG islands in the promoter region of RASSF5A gene in the OSCC tissues and cells may be related to the occurrence and progression of OSCC,and may be an important indicator for evaluating the prognosis of OSCC.