高恶性膀胱移行细胞癌与相应正常上皮差异表达基因的克隆及功能研究
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摘要
膀胱癌的发生发展跟多种基因的突变和表达异常有关，要弄清楚其确切的机制，需要对选择性表达的基因进行分离、克隆和序列分析，并对其氨基酸组成、表达产物和结构功能等进行相应的研究。以往研究基因表达差异的方法很多，但最行之有效的方法是Diatchenko建立的抑制消减杂交技术，它不仅特异性高、灵敏度高、假阳性率低，而且能克隆高、低丰度基因。
本课题应用抑制消减杂交技术构建人膀胱移行细胞癌和正常膀胱上皮组织差异表达的cDNA消减文库，进一步利用斑点印迹杂交(Dot blot)从中筛选出BTCC差异表达的基因，随机选取部分克隆进行测序，结果在GenBank中作同源性对比分析。应用Northern blot技术对所克隆的高拷贝新基因片段进行差异表达分析；对其中有明显差异表达的膀胱癌特异的新基因片段应用SMART RACE技术进行全长克隆，应用免疫荧光原位杂交技术进行染色体定位，并初步推断所克隆的新基因全长序列的编码区和蛋白结构。应用反义核酸技术阻断该新基因在膀胱癌EJ细胞中的表达后，观察膀胱癌EJ细胞形态、生长、凋亡、死亡、转移、增殖活性等的变化，通过以上研究来探讨该新基因的功能。最后应用RT-PCR技术检测新基因在不同分级、分期临床膀胱癌标本中
的表达及意义。
研究结果表明:应用SSH技术构建了具有高消减效率的人膀胧癌
组织cDNA消减文库，文库中含有376个阳性克隆和83个阴性克隆，
阳性克隆率为78%。随机挑出的100个阳性克隆中，89个克隆载有插
入片段，片段插入率达84%，提示每一克隆均有插入特异片段的可能性。
在89个克隆中，有76个克隆含有肿瘤差异表达的基因片段，4个克隆
含有可能污染的基因片段，9个克隆含有未消减掉的来自正常组织的基
因片段，说明SSH具有很高的消减效率。对76个克隆中的插入片段进
行同源性比对分析，其中有66个克隆代表23种已知基因，其余的IC
个克隆代表5种未知基因，这表明消减文库中37%的克隆代表着不同
的基因。在23种差异表达的己知基因中，18种为跟膀肤癌有关的基因，
其余5种基因目前尚无文献报道跟膀肤癌有关，这些差异表达基因的克
隆为膀肤移行细胞癌的进一步研究提供了新的线索，可能从中筛选到
膀肤移行细胞癌新的治疗靶点或瘤标。结合其他已发现的差异表达的
已知基因，绘制膀肤移行细胞癌差异表达的基因谱，以利彻底阐明其
发生、发展的分子生物学机制。
未知新序列中BQ135232共有3个拷贝。Northern杂交分析显示它
在膀肤癌组织中明显表达，而在正常膀胧组织中无明显表达，这表明
BQ135232是膀胧癌特异表达的新基因。应用SMART RACE技术获得
BQI 35232的全长，经GENBANK检索证明其为序列和功能均未知的新基
因。应用免疫荧光原位杂交染色体定位结果显示BQ135232位于第12
号染色体近末端处。组织分布显示BQI 35232在膀脱癌组织和膀肤癌细
胞株中特异性高表达，而在正常膀眺组织表达量极低甚至不表达，在
肾癌、前列腺癌组织及非膀肤癌细胞株中表达则很低，在乳腺癌组织、
j反津医科大学博d匕学位论文
、
肺癌组织、肝癌组织及宫颈癌组织中未见明显的表达。BQ135232反义
寡核昔酸转染膀肮癌EJ细胞后，可明显抑制其生长、降低其增殖活性、
减少核分裂、并诱导其持续性凋亡。应用RT一PCR技术检测BQI 35232
在不同分级、分期膀胧癌标本中的表达，结果表明BQ 1 35232在高级、
高期膀胧癌中的表达明显高于在低级、低期膀肤癌中的表达。本研究
表明BQ135232是跟膀肤癌发生发展密切相关的新基因，它的表达与膀
肤癌细胞的生长、增殖、抗凋亡密切相关。它的成功克隆为阐明膀胧
癌发生发展的分子生物学机制开辟了新的途径，为今后膀胧癌的诊断、
治疗提供了新的理论依据。
The occurrence and development of bladder transitional cell carcinoma (BTCC) is related with the mutation and abnormal expression of many genes. It is necessary that clone and identify BTCC-specially expressed genes to elucidate the mechanism of BTCC. Attempts have been made to isolate genes over expressed in cancer cells with methods such as representational difference analysis (RDA), subtractive hybridization, differential display of mRNA (DD), and differential subtraction display (DSD). Although these methods have been proved to be powerful tools for the construction of gene indexes, they are not excellent enough on account of their low specificity and sensitivity.
Suppression subtractive hybridization technique was used to construct a cDNA subtractive library of human BTCC, Dot blot was then used to screen the genes that differently expressed between BTCC and it's normal epithelia tissues. Some clones in the library were selected
randomly and sequenced. Then the results were analyzed with the Blast
software. The novel gene which had many copies in the library was identified its expression in BTCC and normal epithelia tissues by Northern blot. For the novel gene fragment that was specially expressed in BTCC, we used SMART RACE technology to clone its full length and also used fluorescence immunology situ hybridization to identify its chromosome location. After transfecting the antisense oligodeoxynucleotide of BQ135232 into bladder carcinoma EJ cell line, the proliferation activity, growth speed, apoptosis, and mortality changes in EJ cell were determined. Finally, the expression of BQ135232 in different stages and grades of BTCC tissues were studied by the assay of RT-PCR.
The results showed that human BTCC subtractive library with highly subtractive efficiency was set up successfully. The amplified library contains 376 positive clones and 83 negative clones. Random analysis of 100 clones with restriction enzyme digestion shows that 89 ones contain inserts. After screened by Dot blot, 76 inserts of all the 89 PCR-amplified ones were real positive. Sequencing and analyzing with BLAST, all these 76 inserts represented 23 kinds of known genes and 5 anonymous ESTS in which the fragment of BQ135232 has 3 copies. Northern blot analysis showed that BQ135232 expressed higher in bladder carcinoma tissues or cell lines than other tumors or cell lines. By
using Smart Race technique, we obtained the full length of the novel
gene that was about 1.5kb long. The expression of BQ135232 was higher in higher grades and stages of BTCC than in lower ones. After transfection of BQ135232 antisense oligonucleotide the mortality of EJ cell increased evidently, the proliferation activity and growth speed were inhibited remarkably at the same time. Also the antisense oligonucleotide can induce the apoptosis of EJ cell.
Our research indicated that BQ135232 is an important novel gene related with the generation and development of BTCC. Its over expression could stimulate the growth and proliferation activity of cancer cells and played an anti-apoptosis effect in BTCC. Transfection of antisense oligonucleotide could inhibit the development of BTCC. The study of this gene provided a new clue for research of BTCC, and also provided an instruction for the diagnosis and therapy of BTCC. The construction of highly efficient cDNA subtractive library of human BTCC lays solid foundation for large scale screening and cloning of new and specific oncogenes or tumor suppressor genes of BTCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of BTCC.
本课题应用抑制消减杂交技术构建人膀胱移行细胞癌和正常膀胱上皮组织差异表达的cDNA消减文库，进一步利用斑点印迹杂交(Dot blot)从中筛选出BTCC差异表达的基因，随机选取部分克隆进行测序，结果在GenBank中作同源性对比分析。应用Northern blot技术对所克隆的高拷贝新基因片段进行差异表达分析；对其中有明显差异表达的膀胱癌特异的新基因片段应用SMART RACE技术进行全长克隆，应用免疫荧光原位杂交技术进行染色体定位，并初步推断所克隆的新基因全长序列的编码区和蛋白结构。应用反义核酸技术阻断该新基因在膀胱癌EJ细胞中的表达后，观察膀胱癌EJ细胞形态、生长、凋亡、死亡、转移、增殖活性等的变化，通过以上研究来探讨该新基因的功能。最后应用RT-PCR技术检测新基因在不同分级、分期临床膀胱癌标本中
的表达及意义。
研究结果表明:应用SSH技术构建了具有高消减效率的人膀胧癌
组织cDNA消减文库，文库中含有376个阳性克隆和83个阴性克隆，
阳性克隆率为78%。随机挑出的100个阳性克隆中，89个克隆载有插
入片段，片段插入率达84%，提示每一克隆均有插入特异片段的可能性。
在89个克隆中，有76个克隆含有肿瘤差异表达的基因片段，4个克隆
含有可能污染的基因片段，9个克隆含有未消减掉的来自正常组织的基
因片段，说明SSH具有很高的消减效率。对76个克隆中的插入片段进
行同源性比对分析，其中有66个克隆代表23种已知基因，其余的IC
个克隆代表5种未知基因，这表明消减文库中37%的克隆代表着不同
的基因。在23种差异表达的己知基因中，18种为跟膀肤癌有关的基因，
其余5种基因目前尚无文献报道跟膀肤癌有关，这些差异表达基因的克
隆为膀肤移行细胞癌的进一步研究提供了新的线索，可能从中筛选到
膀肤移行细胞癌新的治疗靶点或瘤标。结合其他已发现的差异表达的
已知基因，绘制膀肤移行细胞癌差异表达的基因谱，以利彻底阐明其
发生、发展的分子生物学机制。
未知新序列中BQ135232共有3个拷贝。Northern杂交分析显示它
在膀肤癌组织中明显表达，而在正常膀胧组织中无明显表达，这表明
BQ135232是膀胧癌特异表达的新基因。应用SMART RACE技术获得
BQI 35232的全长，经GENBANK检索证明其为序列和功能均未知的新基
因。应用免疫荧光原位杂交染色体定位结果显示BQ135232位于第12
号染色体近末端处。组织分布显示BQI 35232在膀脱癌组织和膀肤癌细
胞株中特异性高表达，而在正常膀眺组织表达量极低甚至不表达，在
肾癌、前列腺癌组织及非膀肤癌细胞株中表达则很低，在乳腺癌组织、
j反津医科大学博d匕学位论文
、
肺癌组织、肝癌组织及宫颈癌组织中未见明显的表达。BQ135232反义
寡核昔酸转染膀肮癌EJ细胞后，可明显抑制其生长、降低其增殖活性、
减少核分裂、并诱导其持续性凋亡。应用RT一PCR技术检测BQI 35232
在不同分级、分期膀胧癌标本中的表达，结果表明BQ 1 35232在高级、
高期膀胧癌中的表达明显高于在低级、低期膀肤癌中的表达。本研究
表明BQ135232是跟膀肤癌发生发展密切相关的新基因，它的表达与膀
肤癌细胞的生长、增殖、抗凋亡密切相关。它的成功克隆为阐明膀胧
癌发生发展的分子生物学机制开辟了新的途径，为今后膀胧癌的诊断、
治疗提供了新的理论依据。
The occurrence and development of bladder transitional cell carcinoma (BTCC) is related with the mutation and abnormal expression of many genes. It is necessary that clone and identify BTCC-specially expressed genes to elucidate the mechanism of BTCC. Attempts have been made to isolate genes over expressed in cancer cells with methods such as representational difference analysis (RDA), subtractive hybridization, differential display of mRNA (DD), and differential subtraction display (DSD). Although these methods have been proved to be powerful tools for the construction of gene indexes, they are not excellent enough on account of their low specificity and sensitivity.
Suppression subtractive hybridization technique was used to construct a cDNA subtractive library of human BTCC, Dot blot was then used to screen the genes that differently expressed between BTCC and it's normal epithelia tissues. Some clones in the library were selected
randomly and sequenced. Then the results were analyzed with the Blast
software. The novel gene which had many copies in the library was identified its expression in BTCC and normal epithelia tissues by Northern blot. For the novel gene fragment that was specially expressed in BTCC, we used SMART RACE technology to clone its full length and also used fluorescence immunology situ hybridization to identify its chromosome location. After transfecting the antisense oligodeoxynucleotide of BQ135232 into bladder carcinoma EJ cell line, the proliferation activity, growth speed, apoptosis, and mortality changes in EJ cell were determined. Finally, the expression of BQ135232 in different stages and grades of BTCC tissues were studied by the assay of RT-PCR.
The results showed that human BTCC subtractive library with highly subtractive efficiency was set up successfully. The amplified library contains 376 positive clones and 83 negative clones. Random analysis of 100 clones with restriction enzyme digestion shows that 89 ones contain inserts. After screened by Dot blot, 76 inserts of all the 89 PCR-amplified ones were real positive. Sequencing and analyzing with BLAST, all these 76 inserts represented 23 kinds of known genes and 5 anonymous ESTS in which the fragment of BQ135232 has 3 copies. Northern blot analysis showed that BQ135232 expressed higher in bladder carcinoma tissues or cell lines than other tumors or cell lines. By
using Smart Race technique, we obtained the full length of the novel
gene that was about 1.5kb long. The expression of BQ135232 was higher in higher grades and stages of BTCC than in lower ones. After transfection of BQ135232 antisense oligonucleotide the mortality of EJ cell increased evidently, the proliferation activity and growth speed were inhibited remarkably at the same time. Also the antisense oligonucleotide can induce the apoptosis of EJ cell.
Our research indicated that BQ135232 is an important novel gene related with the generation and development of BTCC. Its over expression could stimulate the growth and proliferation activity of cancer cells and played an anti-apoptosis effect in BTCC. Transfection of antisense oligonucleotide could inhibit the development of BTCC. The study of this gene provided a new clue for research of BTCC, and also provided an instruction for the diagnosis and therapy of BTCC. The construction of highly efficient cDNA subtractive library of human BTCC lays solid foundation for large scale screening and cloning of new and specific oncogenes or tumor suppressor genes of BTCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of BTCC.
引文
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2 Diatchenko L, Lau YFC, Canpbell APR, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probs and libraries. Proc Natl Acid Sci USA, 1996,93:6025-6030．
3 J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.质粒DNA的分离与纯化 [M].见:金冬雁,黎孟枫 翻译.分子克隆实验指南.第二版.北 京:科学出版社,1996: 19-24．
4Hedric SM, Cohen DI, Nielsen EA, Davis MM. Isolation of cDNA clones encording T cell-specific membrane-associated proteins. Nature, 1994, 308: 19-153．
5 Hubank M, Schatz DG. Indetifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res, 1994, 22:5640-5648．
6Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992,257:967-97．
7Chen I, Hsieh T, Thomas T, et al. Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells
using suppression subtractive hybridization [J]. Gene,2001, 262 (1-2) :207-214．
8Miyasaka Y, Enomoto N, Nagayama K, et al. Analysis of differentially expressed genes in human hepatocellular carcinoma using suppression subtractive hybridization [J]. Br J Cancer 2001, 85(2) : 228-234．
9Hufton SE, Moerkerk PT, Brandwijk R, et al. A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization [J]. FEBS Letters, 1999,463 (1-2) : 77-82．
10Urskaya NG,Diatchenko L,Chenchik A,et al. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem, 1996 Aug 15; 240(1) : 90-97．
11Von Stein OD,Thies WG,Hofmann M,et al. A high throughout screeing for rarely transcribed differentialy expressed genes. Nucleic Acid Res, 1997, 25: 2598-2602．
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37 Moriyama M, Akiyama T, Yamamoto T, et al. Expression of c-er-B2 gene product in urinary bladder cancer. J Urol, 1991, 145:423-427．
2 Diatchenko L, Lau YFC, Canpbell APR, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probs and libraries. Proc Natl Acid Sci USA, 1996,93:6025-6030．
3 J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯.质粒DNA的分离与纯化 [M].见:金冬雁,黎孟枫 翻译.分子克隆实验指南.第二版.北 京:科学出版社,1996: 19-24．
4Hedric SM, Cohen DI, Nielsen EA, Davis MM. Isolation of cDNA clones encording T cell-specific membrane-associated proteins. Nature, 1994, 308: 19-153．
5 Hubank M, Schatz DG. Indetifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res, 1994, 22:5640-5648．
6Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992,257:967-97．
7Chen I, Hsieh T, Thomas T, et al. Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells
using suppression subtractive hybridization [J]. Gene,2001, 262 (1-2) :207-214．
8Miyasaka Y, Enomoto N, Nagayama K, et al. Analysis of differentially expressed genes in human hepatocellular carcinoma using suppression subtractive hybridization [J]. Br J Cancer 2001, 85(2) : 228-234．
9Hufton SE, Moerkerk PT, Brandwijk R, et al. A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization [J]. FEBS Letters, 1999,463 (1-2) : 77-82．
10Urskaya NG,Diatchenko L,Chenchik A,et al. Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal Biochem, 1996 Aug 15; 240(1) : 90-97．
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