Ma Ye-Wei, Qian Xin-Lai, Zhao Qing-Zheng, Zhou Xiao-Shan, Li Yan-Chun
Cancer Institute,Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, PR China.
Ai Zheng. 2004 Feb;23(2):146-9.
BACKGROUND & OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug-sensitivity of tumor cells to several antitumor agents, and also increase the radio-sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer-related genes,which were differentially expressed in drug-sensitive tumor cells using suppression subtractive hybridization (SSH).
To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed-gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi-quantitative RT-PCR analysis.
The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over-expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over-expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi-quantitative RT-PCR analysis, and the difference of mRNA expression was approximately 3-8 times.
Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full- length genes and studying their functions.
已有充分证据表明，E1A作为一种肿瘤抑制基因，能够抑制不同肿瘤的生长和转移，并逆转恶性表型。特别是，该基因具有极大增强肿瘤细胞对多种抗肿瘤药物的药物敏感性以及提高放射敏感性的能力。然而，E1A发挥其抗肿瘤功能所涉及的相关基因仍不清楚。本研究的目的是利用抑制性消减杂交（SSH）技术分离在药物敏感肿瘤细胞中差异表达的E1A抗癌相关基因。
分别以经E1A蛋白处理的人淋巴结转移肿瘤细胞（LN686）的mRNA和亲本LN686细胞作为测试方和驱动方，构建人淋巴结转移肿瘤细胞（LN686）的SSH文库。随机挑选文库中的阳性克隆，采用斑点杂交分析差异表达基因片段的表达模式。对cDNA片段序列进行分析，并与GenBank中的序列进行比较。通过半定量RT-PCR分析确定测试方和驱动方中这些新基因的mRNA水平。
SSH文库包含约7000个阳性克隆。用PCR对384个克隆进行随机分析表明，362个克隆含有插入片段。斑点杂交结果表明，与驱动方相比，这些基因在测试方中显著过度表达。对这362个克隆进行测序并进行BLAST分析，结果显示有10个克隆为新的ESTs，并已在GenBank中注册。通过半定量RT-PCR分析，与亲本LN686细胞相比，7个新基因的mRNA水平在经E1A蛋白处理的LN686细胞中过度表达，mRNA表达差异约为3 - 8倍。
通过SSH技术分离出10个新的基因片段，为进一步克隆其全长基因并研究其功能奠定了基础。
