染色体7q32-ter最小共同缺失区鼻咽癌抑瘤基因候选者的筛选

Cloning of a novel putative tumor suppressor gene associated with nasopharyngeal carcinoma on the smallest common deletion region of 7q32-ter

目的：在明确 7q32- ter区域等位基因杂合性丢失最小共同缺失区位于以 D7S509为中心的 D7S500- D7S509- D7S495的基础上，筛选和克隆位于该区内鼻咽癌相关的候选抑瘤基因。方法：应用定位—候选克隆策略筛选位于该最小共同缺失区的 3′末端 ESTs(expressed sequence tags,ESTs),RT- PCR和 Northern杂交筛选出在鼻咽癌细胞株和活检组织中表达下调的 ESTs,采用 cDNA克隆测序和生物信息学资源获取候选 EST的全长 cDNA。 Southern杂交和甲基化分析研究候选基因表达下调的机制。结果：筛选 D7S500- D7S495区域内的 12个代表新基因的 3′末端 ESTs序列，发现一个 EST（ AI290024）在鼻咽癌细胞株及 50％（ 6/12）的鼻咽癌活检组织中表达下调， Multiple Tissue Northern(MTNTM) Blot杂交结果显示该 EST在心、肝、肾等多种组织中存在大小 2.0 kb的转录本。通过测定包含该 EST的 cDNA克隆，获得该新基因（命名为 NAG- 22基因） 2.3 kb转录本的全长 cDNA(GenBank登录号： AF247820),编码 77AA，含有 disintegrin的结构域。 NAG22在 NPC中表达下调的机制不是高甲基化和基因拷贝数的丢失。结论： NAG22是定位于 7q32- ter最小共同缺失区的一个与鼻咽癌相关的抑瘤基因候选者。

Objective： The current study was designed to clone novel putative tumor suppressor genes associated with NPC on the smallest common deletion region of 7q32- ter. Method： the expression of 3′ end expressed sequence tags(ESTs) localized within this smallest common deletion region were investigated in NPC cell line HNE1, primarily cultured nasopharyngeal epithelial cells and NPC biopsies using differential RT- PCR and Northern hybridization. The full- length cDNA of candidated EST was cloned through cDNA clone sequencing and bioinformatics.Southern Blot and methylation analysis studied the machanism of down- expression of NAG22 in NPC. Results： one EST(AI290024)was down- expressed in HNE1 and 50％ of NPC biopsies. Multiple Tissue Northern Blot showed this EST has the transcript of size of 2.0 kb in human heart, liver, kidney and other tissues. A novel gene(named NAG22) with a full- length cDNA of 2.3 kb (GenBank No.AF247820)was cloned through DNA sequencing. Bioinformatics analyzed this gene encoded 77 AA and contained the domain of disintegrin.Methylation and loss of gene copies were not the machanism of the down- expression of NAG22. Conclusion： the new NAG22 located in this region may be a putative tumor suppressor gene associated with NPC.