一个定位于7q31-32的鼻咽癌负相关新基因的初步研究

Preliminary Study of a Novel Gene Negatively Associated with Nasopharyngeal Carcinoma on 7q31-32

目的：克隆染色体7q31-32区域D7S495附近鼻咽癌相关的候选抑瘤基因，并研究其在鼻咽癌发生发展中的作用。方法：应用定位－候选克隆策略结合生物信息学手段筛选鼻咽癌负相关的EST(Expressed Sequence Tags)，cDNA克隆测序和5′RACE扩增获取候选EST的cDNA序列。Northern杂交和RT-PCR检测其组织表达谱及其在正常人胚鼻咽上皮与鼻咽癌细胞株和活检组织中的表达差异。Southern杂交和甲基化分析表达差异的机制。结果：克隆到一个位于7q31-32的与鼻咽癌负相关的新基因(GenBank登录号：AF196976，命名为NAG-14)，该基因编码649AA，含有LRR和IgC2结构域。Multiple Tissue Northern(MTNTM) Blot杂交结果显示该基因在脑组织中表达较强，而在心、肝、肾、肺、脾、骨骼肌和胎盘等多种组织中检测不到表达。RT-PCR检测发现其在鼻咽癌细胞株和75％活检组织表达缺失或下调，Southern 和甲基化分析表明其在鼻咽癌细胞株中某些位点发生甲基化修饰，鼻咽癌活检组织中存在遗传物质丢失。结论：NAG_14可能是定位于7q31-32的一个与鼻咽癌相关的抑瘤基因候选者。

Objective：This study was designed to clone a novel candidate for tumor suppressor gene associated with nasopharyngeal carcinoma(NPC) on chromosome 7q31-32 and to investigate its function in carcinogenesis. Methods：Combined with positional-candidate strategy, the candidate EST related to NPC was screened by Bioinformatics. The cDNA of candidate EST was obtained through cDNA clone sequencing and 5′rapid amplification of cDNA end(RACE). Tissue distribution and expression difference between primarily cultured nasopharyngeal epithelial cells and NPC biopsies were examined by Northern blot analysis and RT-PCR. The function in carcinogenesis of NPC was examined by Southern blot and methylation analysis. Results：A novel gene(named NAG_14) with partial cDNA sequence of 2.3 kb (Genbank accession number：AF196976) was cloned through cDNA sequencing combined with 5′RACE. The gene contained a complete 1950 bp open reading frame(ORF), which encoded 649AA and contained leucine rich repeats (LRRs),one transmembrane region, IgC2 domain and signal peptide by Bioinformatics analysis. Northern blot analysis showed the gene had strong expression in brain, while no expression in other seven normal human tissues. The gene had no or much lower expression in NPC cell line HNE1 and NPC biopsies (15/20) compared with primarily cultured nasopharyngeal epithelial cells using differential RT-PCR. Methylation examination and Southern blot suggested high methylation and loss of genetic substance may be the mechanism of the down-expression of NAG_14 in NPC. Conclusion：The new gene, NAG_14, located in chromosome 7q31-32 may be a tumor suppressor gene candidate associated with NPC. The following study will focus on the full-length cDNA cloning and deep function study of NAG_14.