喉鳞癌Apaf-1基因表达及启动子区甲基化研究

Expression and Promoter Methylation of Apaf-1 Gene in Laryngeal Squamous Cell Carcinoma

喉鳞癌细胞存在多种癌基因和抑癌基因异常 ,Apaf 1(apoptoticproteaseactivatingfactor 1)基因是诱导细胞凋亡的肿瘤抑制基因。为探讨Apaf 1基因在喉鳞癌发生中的作用 ,应用半定量PCR方法分析Apaf 1的表达用比较基因组杂交 (ComparativeGenomicHybrodization ,CGH)和杂合性丢失 (LossofHeterozygosity ,LOH)分析方法对喉鳞癌病人Apaf 1基因所在的 12 q2 2 2 3区域缺失情况进行研究 ,并用甲基化特异PCR对该基因启动子区甲基化情况进行了分析。结果表明 :11例喉鳞癌组织出现Apaf 1mRNA表达明显下调 ,占 4 0 7% (11/ 2 7) ,而 6例良性喉肿瘤未发现缺失或下调 ;CGH分析发现 ,18例喉鳞癌仅发现 2例存在 12 q2 2 2 3区域缺失 ,未发现扩增 ;LOH分析发现 ,72例喉癌组织Apaf 1基因的 5个多态位点LOH发生频率低 ,分别为 18 2 % (D12S346 )、13 9%(D12S170 6 )、18 2 % (D12S32 7)、2 2 2 % (D12S16 5 7)和 16 6 % (D12S393) ,11例出现Apaf 1mRNA下调的喉癌组织均检测到启动子区甲基化 ,而 16例Apaf 1mRNA表达未下调者仅 1例有甲基化 ,两者有显著差异 (χ2 检验 ,P=0 0 0 0 1)。通过以上结果 ,首次证实Apaf 1基因与喉鳞癌相关 ,在喉鳞癌中Apaf 1基因缺失发生率低 。

Loss of tumor suppressor genes and apoptosis-associated genes was common event in laryngeal squamous cell carcinoma (LSCC).Apaf-1(apoptotic protease activating factor-1)is a key factor in cytochrome C-dependent apoptotic pathway.To investigate the effect of Apaf-1 in progression of LSCC,we analyzed Apaf-1 from DNA and RNA levels.Semi-quantitative RT-PCR analysis of Apaf-1 mRNA showed that over 40 percent of LSCCs(11/27)were low expression compared to the para-neoplastic laryngeal tissues(PNTs).The results of CGH indicated loss in 2 of 18 cases but no amplification on chromosome 12q22-23.The LOH frequencies of D12S327,D12S1657,D12S393,D12S1706,D12S346 on 12q22-23 were 18.2%,13.6%,18.2%,22.2% and 16.6%,respectively in 72 matched samples of LSCCs and PNTs.Methylation-specific PCR displayed that all of 11 LSCCs,whose expression of Apaf-1 mRNA down-regulated,were methylated in promoter regions.In contrast,only 1 of 16 LSCCs with no changes in Apaf-1 mRNA levels was methylated(χ~2 test,P=0.0001).The results implied that abnormal expression of Apaf-1 participates in the genesis of LSCCs and decreased expression of Apaf-1 mRNA were not mainly due to the deletion of Apaf-1 gene.The Apaf-1 DNA methylation in promoter region might contribute to the decreased transcription of Apaf-1 in LSCCs.