应用抑制消减杂交技术筛选人肾癌差异表达新基因

Screening of Novel Genes Differentially Expressed in Human Renal Cell Carcinoma by Suppression Subtractive Hybridization

背景与目的:认识肾癌差异表达基因有助于阐明肾癌发生、发展的分子机制,但至今有关肾癌差异基因尤其肾癌特异相关基因的研究仍不令人满意。本实验应用抑制消减杂交技术筛选人肾癌组织与正常肾组织间差异表达的新基因,以期克隆出新的肾癌特异相关基因。方法:以肾癌组织mRNA为检测对象(Tester),正常肾组织mRNA为驱赶者(Driver),构建cDNA消减文库。随机挑取文库克隆进行酶切及测序,所得结果在GenBank中做同源性比对分析,对感兴趣的片段进行电子定位确定其在染色体的位置。用Northernblot、半定量RT-PCR方法检测新基因在肾癌组织与正常肾组织中的差异表达。结果:文库包含414个阳性克隆;随机挑取280个克隆提取质粒并酶切分析,其中265个有插入片段;将其中80个克隆进行测序,初步显示28、158、170、249号4个克隆为新基因片段,电子定位表明上述4个基因分别位于染色体21q22、4p15.3、9q34、22q11.2,已在GenBank中登录(BM181083,BI784487,BI863835,BI863386)。对其中28、170号克隆用Northern杂交、半定量RT-PCR检测,证实新基因在肾癌组织中表达较正常肾组织显著增高。结论:抑制消减杂交技术是筛选、克隆肾癌差异表达新基因的有效手段;筛选到的新基因片段为进一步克隆其全长、研究基因功能提供了实验基础。

Background and Objective: Identifying the differentially expressed genes in renal cell carcinoma (RCC) contributes to the elucidation of its genetic basis. However,the above knowledge has not yet been fully understood. The aims of this experiment were to screen novel genes differentially expressed in RCC tissues by suppression subtractive hybridization (SSH) and clone RCC specific related genes. Methods: To construct SSH library of RCC by using the mRNA from RCC tissues and matched normal kidney tissues as tester and driver, respectively. Partial positive clones in the library were selected randomly and sequenced, then analyze the sequences with the BLAST software. To confirm the location of the fragments of interest in human chromosome through comparing their sequences with the human genome draft. mRNA levels of the novel genes in RCC and matched normal kidney tissues were determined by Northern blot and semi quantitative RT PCR analysis. Results: The SSH library contained 414 positive clones. Random analysis of 280 clones with enzyme restriction showed that 265 clones contained cDNA fragments distributed mainly between 300 900bp. Among 80 arbitrary clones which were derived from above 265 clones and sequenced, No.28 ,158,170,and 249 clones are previously unknown genes and located in human chromosome 21q22,4p15.3,9q34,and 22q11.2 by electronic mapping, respectively. The consequence of semi quantitative RT PCR demonstrated that mRNA levels of the two novel genes were overexpressed in RCC compared to matched normal tissues by more than 2 6 folds. Northern blot analysis confirmed the above results. Conclusions: SSH is a reliable strategy for screening novel genes differentially expressed in RCC. The novel gene fragments can be used to clone their full length and further to study their functions.