Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridi-zation and cDNA library array  

作　　者：AI Junkui1, ZHANG Zhiwen2, XIN Dianqi1, ZHU Hongjian1, YAN Quanjian3, XIN Zhongcheng1, NA Yanqun1 & GUO Yinglu1 1. Institute of Urology, Peking University & Department of Urology, First Hospital, Peking University, Beijing 100034, China 2. Department of Physiology, Peking University Health Science Center, Beijing 100083, China 3. Department of Urology, Xijing Hospital, the 4th Military Medical University, Xi抋n 710033, China 

出　　处：《Science China(Life Sciences)》2004年第2期148-157,共10页中国科学（生命科学英文版）

基　　金：co-supported by the Chinese Capital Scientific Development Fund for Medicine(ZD199803);Genomic Research Fund for Human Disease of Peking University.

摘　　要：To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon mem-brane and the over-expressed genes were then screened by hybridizing the filter with radioac-tively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank da-tabase. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular en-dothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohis-tochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon mem-brane and the over-expressed genes were then screened by hybridizing the filter with radioac-tively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank da-tabase. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular en-dothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohis-tochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.

关 键 词：SUPPRESSION subtractive HYBRIDIZATION (SSH)  cDNA microarray  RENAL cell carcinoma (RCC)  gene  differential expression. 

分 类 号：R737.11[医药卫生—肿瘤]