P27基因甲基化在鼻咽癌发生发展中的作用

Role of methylation of P27 gene in pathogenesis of nasopharyngeal carcinoma

目的探讨P27基因甲基化在鼻咽癌发生发展中的作用。方法运用甲基化特异性PCR技术对5个鼻咽癌细胞株(CNE1、CNE2、TWO3、HNE1、HONE1)和1个正常鼻咽上皮细胞株(NP69),54例鼻咽癌组织和20例正常鼻咽上皮组织的P27基因启动子区甲基化状态进行检测。半定量反转录PCR法检测加入甲基转移酶抑制剂5-氮-2-脱氧胞苷(5-aza-2-deoxycytidine)后CNE1和CNE2中P27基因转录表达的变化,分析鼻咽癌组织中P27基因甲基化与临床因素的关系以及去甲基化对P27基因转录表达的影响。结果正常鼻咽上皮细胞株NP69及20例正常鼻咽上皮组织中均未检测到P27基因甲基化;5个鼻咽癌细胞株中P27基因甲基化频率为80%(4/5),54例鼻咽癌组织中P27基因甲基化频率为63%(34/54),二者与正常鼻咽上皮细胞株及正常鼻咽上皮组织比较差异均有统计学意义(P<0.01),进一步分析发现P27基因甲基化状态与患者年龄、性别、T分期、N分期、TNM分期、病理类型等临床因素均无相关性(P>0.05),经5-氮-2-脱氧胞苷处理后CNE1和CNE2中P27基因的转录表达增加。结论P27基因甲基化与其转录表达密切相关,是基因表达调节的一种重要方式。P27基因甲基化具有肿瘤特异性,为鼻咽癌早期事件,有望成为早期分子生物学辅助诊断的标志物,并有可能作为去甲基化基因治疗的靶点。

Objective To assess the role of methylated P27 gene in the tumorigenesis and development of nasopharyngeal carcinoma （NPC）.MethodsThe methylation-specific PCR （MSP） was used to detect methylation status of P27 gene in five NPC cell lines （CNE1,CNE2,TWO3,HNE1,HONE1） and one normal nasopharyngeal epithelia cell line （NP69）,54 specimens of NPC tissues and 20 specimens of normal nasopharyngeal epithelia （NNE） tissues.mRNA expression of p27 gene in two NPC cell lines （CNE1 and CNE2） were analyzed before and after treatment with the methyltransferase inhibitor 5-aza-2-deoxycytidine.In addition,alterations of mRNA expression of P27 gene were detected by semi-quantitative reverse transcription PCR （RT-PCR）.The relationship between promoter methylation and clinical factors,demethylation and mRNA expression of the gene was analyzed.ResultsPromoter methylation of P27 was detected in 80% （4/5） of NPC cell lines and 63% （34/54） of primary tumors,but was not in the normal nasopharyngeal epithelia cell line （NP69） and any of 20 cases of normal nasopharyngeal epithelial tissue.The methylation of P27 was significantly statistically different between NPC cell lines and NPC primary tumors and NP69 cell line and normal nasopharyngeal epithelia tissues （P0.01）.There was no significant difference between promoter methylation and other clinical factors （P0.05）.After 5-aza-2-deoxycytidine treatment,the expression level of mRNA elevated in CNE1 and CNE2.ConclusionsThe close correlation between P27 methylation and low mRNA expression suggests that methylation is an important pathway in regulating gene expression.The promoter methylation of P27 gene has high specificity in distinguishing cancers from normal tissues,and is an early event in NPC.It may provide an evidence for early molecular diagnosis and potential therapeutic target of NPC.