人鼻咽癌中差异表达cDNA序列的cDNA代表性差异分析

cDNA representational difference analysis of differentially expressed cDNA sequences in human nasopharyngeal carcinoma.

作者信息

Zhan F, Cao L, Bin L, Jiang N, Deng L, Xie Y, Tan G, Li G

机构信息

Cancer Research Institute, Hunan Medical University, Changsha 410078, China.

出版信息

Chin Med J (Engl). 1999 Jun;112(6):538-42.

PMID:11601334

Abstract

OBJECTIVE

To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes.

METHODS

Representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE1. The source of differentially expressed products were proved by Southern blot, Northern blot and in situ hybridization. The fragments were cloned with pGEM-T easy kit and sequenced by the chain termination reaction.

RESULTS

Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down-regulated in the NPC HNE1 cells. Some of the genes were expressed only in human nasopharyngeal epithelial cells but deleted or down-regulated in the biopsies of NPC. Of these obtained clones, some were the sequences of the human known genes including house-keeping genes, the others represented novel gene sequences.

CONCLUSION

The differentially expressed products including the candidates of tumor-suppressor genes may be associated with the initiation of the NPC.

摘要

目的

寻找与人类鼻咽癌（NPC）发病机制相关的差异表达序列，包括肿瘤抑制基因的候选序列。

方法

采用代表性差异分析（RDA）技术，分离正常人鼻咽上皮细胞原代培养物cDNA与NPC细胞系HNE1 cDNA之间的差异表达序列。通过Southern印迹、Northern印迹和原位杂交对差异表达产物的来源进行验证。用pGEM-T easy试剂盒克隆片段，并通过链终止反应进行测序。

结果

以正常人鼻咽上皮细胞cDNA为检测扩增子，NPC细胞系HNE1 cDNA为驱动扩增子，经cDNA RDA在第四次消减杂交中分离出4个差异表达的cDNA片段。这些差异cDNA片段表明它们确实来自检测扩增子，在NPC HNE1细胞中未表达或下调。其中一些基因仅在人鼻咽上皮细胞中表达，但在NPC活检组织中缺失或下调。在这些获得的克隆中，一些是人类已知基因的序列，包括管家基因，其他则代表新的基因序列。