一个新鼻咽癌抑瘤候选基因的克隆及其功能初步分析

Molecular Cloning and Functional Primary Study of a Novel Candidate Tumor Suppressor Gene Related with Nasopharyngeal Carcinoma.

从cDNA代表差异分析法 (cDNArepresentationaldifferenceanalysis ,cDNARDA)分离的新cDNA片段入手 ,进一步采用RT PCR验证 ,其中发现AF152 60 5片段在 74 %鼻咽癌 (nasopharyngealcarcinoma ,NPC)活检组织中表达下调和缺失 ,DNA印迹显示其代表一转录本为 2 1kb的基因 ,结合生物信息学 ,运用文库筛选克隆该基因 ,命名为NAG4基因 ,GenBank登录号AF1792 85,定位于 6q2 2 1～ 2 2 33 ,至少含有两个外显子 ,并在第一外显子的上游有TATA盒样序列 ,编码一个 50 8个氨基酸组成的、分子质量为 57 4ku的蛋白质 ;功能预测NAG4基因产物与小鼠溴区蛋白BP75有 84 %同源 ,是含有多个磷酸化位点和溴区结构域的核内转录因子 ;突变分析表明NAG4基因在HeLa细胞株中发生整码突变 .以上结果说明NAG4基因是鼻咽癌抑瘤基因的良好候选者 。

In order to further identify expressing difference of cDNA fragments isolated from cDNA representational difference analysis (cDNA RDA) in nasopharyngeal carcinoma(NPC) biopsies and to clone those deregulated genes related with NPC, reverse transcription polymerase chain reaction ( RT PCR ) was used to identify the differentially expressing of cDNA fragment, among which AF152605 was found to be down regulated in sporadic NPC samples (74%). As it were shown by Northern blot, AF152605 is highly expressed in heart,brain, skeletal muscle and placenta whose transcription size is 2 1 kb. Combining bio informatics , the gene (GenBank accession number AF179285) named as NAG4, was cloned by using library screening, which locates at 6p22～6p23 3. Comparative analysis shows that the NAG4 gene has at least two exons and a “TATAA BOX” sequence lies at upstream of the first exon. And the NAG4 gene encodes a protein proposed of 508 amino acid with predicted molecular mass of 57 4 ku, Blast homology searches reveal that NAG4 protein, containing a bromodomain and several important phosphorylation sites, takes on high similarity to musculus bromodimain containing protein BP75. Moreover, condon mutation of NAG4 gene was found in HeLa cell line. So it is further demonstrated that NAG4 gene is a good candidate of putative tumor suppressor genes associated with NPC, whose down expression may be involved in the development of NPC.