p21和p53基因甲基化在喉鳞状细胞癌中的差异化表达及生物学意义

Differentially expression p21 and p53 in laryngeal squamous cell carcinoma

目的:研究p21、p53基因甲基化在喉癌组织和喉癌细胞系Hep-2中的机制。方法:用甲基化特异性PCR检测46例配对的喉癌组织和癌旁组织中p21、p53基因甲基化情况;用MTT法检测去甲基化药物5-azacytidine及作用时间对喉癌细胞系Hep-2增殖的影响;用TUNEL法检测5-azacytidine及作用时间对喉癌细胞系Hep-2凋亡的影响。同时,采用RT-PCR法检测干预前后p21基因mRNA的差异,并采用MSP法检测干预前后p21基因启动子区CpG岛甲基化状态变化。结果:喉癌组织、癌旁组织中p21基因启动子区CpG岛甲基化率分别为45.7%和10.9%,两者比较差异有统计学意义(P<0.05);喉癌组织、癌旁组织中p53基因甲基化率分别为6.5%和4.3%,两者比较差异无统计学意义(P>0.05)。5-azacytidine对Hep-2细胞增殖的抑制作用随着时间的延长逐渐增加,呈时间依赖性;同时,随着天数的增加,细胞凋亡率增加。Hep-2细胞在5-azacytidine干预后p21基因被激活,表达明显增高。喉癌细胞系Hep-2中p21基因甲基化程度为43.13%±0.79%,经5-azacytidine干预后,甲基化程度明显下降,甚至表现为无甲基化。结论:喉癌组织和喉癌细胞系Hep-2中存在p21基因高甲基化,是喉癌区别于正常组织的分子事件。p53基因在喉癌组织和喉癌细胞系Hep-2中低甲基化,其基因的失活可能通过基因突变等其他形式来发生。去甲基化药物5-azacytidine可以通过诱导p21基因启动子CpG岛去甲基化修饰,改变肿瘤细胞细胞周期基因的调控,促进肿瘤细胞凋亡。

Objective：To investigate the role of the methylation of p21 and p53gene in laryngeal cancer and laryngeal cancer cell line Hep-2.Method：Methylation of p21 and p53gene were detected by methylation specific PCR（MSP）in 46 cases with paired cancer tissues and adjacent tissues.Hep-2cell line was treated with 5-azacytidine at different time points,and Hep-2cell growth was detected using the MTT assay.Hep-2cell line was treated with5-azacytidine at different time points,and then apoptosis was detected by the TUNEL assay.The expression difference of p21 mRNA was detected by RT-PCR method before and after intervention.p21 promoter CpG island methylation status was tested by MSP before and after intervention.Result：The incidence was 45.7% of p21 gene promoter CpG island methylation in 46 cases of laryngeal cancer,while methylation rate in adjacent tissues was10.9%.There were significant differences between cancer tissues and adjacent tissues（P〈0.05）.The incidence was 6.5% of p53 gene promoter CpG island methylation in 46 cases of laryngeal cancer,while methylation rate was 4.3%in adjacent tissues,no significant difference of p53 gene methylation was found in laryngeal cancer and counterparts adjacent tissues（P〉0.05）.The effect of Hep-2proliferation inhibition interfered by 5-azacytidine was gradually increased over time in a time-dependent manner.Meanwhile,interfered by 5-azacytidine increased the apoptosis in a time-dependent manner.After interference by 5-azacytidine,p21 gene is activated.The expression of p21 mRNA was significantly higher.The p21 gene methylation degree in laryngeal cancer cell line Hep-2was 43.13%±0.79%.After 5-azacytidine intervention,the degree of methylation was obviously decreased and even showed unmethylation.Conclusion：There was p21 gene promoter CpG island hypermethylation in laryngealcancer and laryngeal cancer cell line Hep-2,which was a molecular event distinguished laryngeal cancer from normal laryngeal tissue.There was p53 gene promoter CpG island hypomethylation in laryngeal cancer and laryngeal cancer cell line Hep-2.Inactivation of the gene may occur by gene mutation forms and others.Demethylation of drug 5-azacytidine can induce p21 gene promoter CpG island demethylation modification,change the regulation of tumor cell cycle genes in cells and promote tumor cell apoptosis.