精细定位和克隆9p21-22区域内鼻咽癌候选抑瘤基因

Refined localization and cloning of a novel putative tumor suppressor gene associated with nasopharyngeal carcinoma on chromosome 9p21-22

目的：进一步精细限定鼻咽癌９ｐ２１－２２区域等位基因杂合性丢失的频率和范围，筛选和克隆其共同缺失区内鼻咽癌相关的候选抑瘤基因。方法：应用１１个定位于 ９ｐ２１－２２区域的高密度微卫星位点，检测 ２５例低分化鼻咽癌患者的杂合性丢失，确定其共同缺失区；用ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ筛出在鼻咽癌细胞株ＨＮＥ１和鼻咽癌活检组织中表达下调的、定位于共同缺失区内的 ３’末端ＥＳＴｓ（Ｅｘｐｒｅｓｓ Ｓｅｑｕｅｎｃｅ Ｔａｇｓ）；采用 ＲＡＣＥ技术和生物信息学资源克隆出候选ＥＳＴ的全长ｃＤＮＡ。结果： ２５例患者中有１７例（６８％）存在一个或多个位点的杂合性丢失，其中Ｄ９ｓ１６１（３５．０％，７／２０），Ｄ９Ｓ１６７８（３１．５％，６／１９），Ｄ９Ｓ２６３（３．３％，６／１８）和Ｄ９Ｓ１８５３（３３．３％，７／２１）四个紧邻位点的丢失频率相对较高，并发现六位患者在该四个位点表现为连续性缺失；筛选Ｄ９Ｓ１６１－Ｄ９Ｓ１８５３区域内２５个代表新基因的３’末端ＥＳＴｓ序列，发现一个ＥＳＴ（ｄｂＥＳＴ：２０８８２５）在鼻咽癌细胞株ＨＮＥ１及７３％（１１／１５）的活检组织中表达降低， Ｍｕｌｔｉｐｌｅ Ｔｉｓｓｕｅ Ｎｏｒｔ

Objectives: To further refine the extent of deletion on chromosome gp21-22 in nasopharyngeal carcinoma (NPC) and clone a new putative tumor suppressor gene associated with NPC. Methods: Loss of heterozygosity (LOH) on chromosome 9p21 -22 was analyzed in 25 paired blood and tumor samples by using 11 high-density microsatellite polymorphic markers; the expression of 3' end express sequence tags (ESTs) localized with in common deletion region was investigated tri NPC cell line HNE1, primary culture nasopharyngeal epithelial cells and BPC biopsies by using differential RT-PCR and Northern hybridization. Rapid amplification of cDNA ends (RACE) and bioinformatic data were used to clone putative EST, full-length cDNA. Results: Seventeen of 25 cases (68%) showed LOH at one or more loci. Higher frequencies of LOH were found at four loci: D9S161 (35. 0% ), D9S1678 (31.5% ), D9S263 (33. 3% ) and D9S1853 (33.3% ), where 6 cases had a contiguous stretch of allelic loss; 25 ESTs localized within D9S161 - D9S1853 were investigated and one EST (dbESI: 208825) was downexpressed in NPC cell line HNE1 and in 73% (11 / 15) NPC biopsies. Multiple tissue blot revealed that this putative EST hybridized to two different size transcripts (approximately 2.4 kb) and 3.5 kb) in fetal heart and brain tissues. A near full-length cDNA of 2.4 kb transcript of this EST was cloned(named NAG-6 gene) and there is no significant homology with any known genes. Conclusions: The minimal common region of deletion might he defined between D9S161 and D9S1853 (estimated about 2.7 cM in extent) at 9p21.1, the new gene NAG-6 located in this region may be a putative tumor suppressor gene associated with NPC.