肝癌患者p16基因甲基化与DNMTs表达相关性分析

Relationgship between expression of DNMTs and p16 methylation in hepatocellular carcinoma

目的:研究p16基因启动子区域异常甲基化所导致的基因表达异常在肝癌形成过程中的作用,探讨p16基因甲基化与DNMTs(DNMT1、DNMT2、DNMT3A和DNMT3B)表达之间的相关性。方法:检测肝癌患者癌组织、癌旁组织和肝硬化组织中p16基因的异常甲基化状态及p16、DNMTs基因mRNA的表达水平;采用甲基化特异性PCR技术检测甲基化状态,荧光定量技术检测mRNA的表达。结果:p16基因在肝癌组织、肝硬化组织和癌旁组织中的甲基化率分别为70.5%(31/44)、37.1%(13/35)和9.1%(4/44),肝癌组织和肝硬化组织中的甲基化改变与癌旁组织比较差异有统计学意义,P<0.01。31例甲基化阳性组织中17例p16基因表达降低或缺失,13例甲基化阴性组织中2例基因表达降低或缺失,甲基化阳性与阴性的p16基因表达水平存在明显差异。癌组织和肝硬化组织中4种DNMT mRNA水平均高于相应癌旁组织。其中DNMT1、DNMT3A和DNMT3BmRNA的表达水平差异有统计学意义,DNMT1:P值分别为0.009和0.020;DNMT3A:P值分别为0.005和0.010;DNMT3B:P值分别为0.039和0.036。DNMT2mRNA的表达水平虽然高于相应癌旁组织,但差异无统计学意义,P值分别为0.120和0.350。DNMT1、DNMT 3A和DNMT3B的表达与p16甲基化有相关性,P值分别为0.013、0.025和0.041。结论:p16基因甲基化是肝癌的早期、频发事件,其改变是其基因表达下降甚至失活的重要原因。DNMTs活性的改变可能促进p16基因的异常甲基化。

OBJECTIVE：To investigat whether p16 transcription was regulated by promoter CpG island hypermethy- lation in hepatocellular carcinoma（HCC）, analysis the relationship between expression of DNMTs（DNMT1, DNMT3A, DNMT3B） and p16 methylation. METHODS： The cancerous,cirrhotic and adjacent tissues were subjected to analysis for p16 promoter CpG island methylation and mRNA transcription, p16 promoter CpG island methylation levels were deter- mined using the methylation-specific PCR and mRNA transcription levels of the gene were detected by quantitative real- time polymerase chain reaction. RESULTS： Methylation was found in cancerous,cirrhotic and adjacent tissues and methyl- ation frequency were 70.5 %（31/44）, 37.1 % （13/35） and 9.1 % （4/44）. There were significantly coincident in cancerous, cirrhotic and adjacent tissues （P〈0.01） ;The level of mRNA for DNMT1 was elevated in cancerous and cirrhotic tissues compared with the level in corresponding adjacent tissues, the P values were 0. 009 and 0. 020 respectively. The expression of DNMT3A and DNMT3B were elevated in cancerous and cirrhotic tissues compared with the level in the corresponding adjacent tissues,DNMT3A： the P values were 0. 005 and 0. 010 respectively. DNMT3B： the P values were 0. 039 and 0. 036 respectively. DNMT2 ：the P values were 0. 120 and 0. 350 respectively. Correlations analysis showed that there were significant associations between overexpression of DNMTs （DNMT1, DNMT3A, DNMT3B） and p16 methylation, the P values were 0. 013,0. 025 and 0. 041 respectively. CONCLUSIONS： p l 6 gene methylation is an early and frequent epige- netie event in the tumor development. The methylation alterations of p16 can contribute to reducing or losing expression of p16 gene,which play an important role in the development of HCC. The overexpression of DNMTs indirectly involve in the process of tumor-suppressor genes methylation.