一个定位于染色体3p25.3的新基因及在鼻咽癌中的表达分析

Molecular cloning of a novel gene located on chromosome 3p25.3 and an analysis of its expression in nasopharyngeal carcinoma

目的 分离位于染色体 3p2 4- 2 6区域中鼻咽癌相关的新基因。方法 在染色体 3p2 4- 2 6鼻咽癌杂合性丢失 (loss of heterozygosity,L OH)高频区 ,用 RT- PCR及 Northern杂交 ,检测了 2 0个表达序列标记 (expressed sequence tag,EST)在鼻咽癌细胞株 HNE1和原代培养的正常鼻咽上皮细胞中的表达水平 ,并对其中一个在鼻咽癌细胞株 HNE1中表达下调的 EST,在 7个正常鼻咽活检组织和 19例鼻咽癌活检组织中的表达进行了检测。用 c DNA文库筛选方法获得其全长 c DNA序列 ,对该序列进行了初步分析。结果 获得了一个位于染色体 3p2 5 .3的新基因 ,命名为 NAG- 7基因 (Gen Bank收录编号 :AF0 86 70 9)。该基因在 2 6 .3% (5 / 19)的鼻咽癌活检组织中表达下调 ,全长 16 6 7bp,其开放阅读框 (open reading frame,ORF)编码一个含 94个氨基酸的碱性蛋白质 ,相对分子质量为 110 2 3870 ,预测为一跨膜蛋白质。它含有一个蛋白激酶 C磷酸化位点和肉豆蔻基位点。迄今为止 ,NAG- 7基因序列在基因库中未见任何同源性基因。结论 NAG- 7基因是一个在鼻咽癌中表达下调的新基因 ,它的改变可能参与了鼻咽癌的发生发展。

Objective To obtain the novel genes associated with human nasopharyngeal carcinoma(NPC) on chromosome 3p24 26. Methods Twenty epithelial derived expressed sequence tags(EST) were selected from chromosome 3p24 26 where loss of heterozygosity(LOH) frequently occurs in NPC tissues. Primers were designed based on the sequences of these ESTs. RT PCR was used to amply their corresponding cDNA fragments from NPC cell line HNE1 and primary cultures of normal nasopharyngeal epithelial cells. The differential expression of two ESTs, T93093 and R41598, was confirmed by Northern blot. Then, expression of EST T93093 was further detected in 7 normal nasopharyngeal and 19 NPC biopsies. cDNA library screening was used to get its full cDNA sequence and the sequence of this novel gene was analyzed by bioinformatics. Results Thirteen ESTs(T62511, N39155, N68660, R61275, T95314, R06143, H52697, H66521, AA128685, AA284537, N52379, AA054180, and H98090) showed the similar expression level and 5 ESTs(R00732, R07573, R98052, H91759, H17566) showed no expression in both types of cells. EST T93093 was down expressed, whereas EST R41598 up expressed in NPC HNE1 cells. The EST T93093 was also found to be down expressed in 26.3%(5/19) of NPC biopsies. The full length cDNA of this gene was obtained and named NAG 7, which is located at chromosome 3p25.3. Its 1677 bp full length cDNA has a potential open reading frame(ORF) predicting a 94 amino acid protein with a molecular weight of 11023.87 Dalton. Bioinformatics analysis of the NAG 7 gene shows that it is a transmembrane protein containing a protein kinase C(PKC) phosphorylation site and a myristyl site. It has no significant homology to any reported genes in database of GenBank(AF086709). Conclusion NAG 7 is a novel gene down expressed in NPC, which may be involved in the development of NPC.