RDA技术分析病毒性肝硬化、原发性肝细胞癌相关基因的研究
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摘要
乙型和丙型肝炎病毒(Hepatitis B／C Virus，HBV／HCV)是引起肝硬化、原发性肝细胞癌等疾病的主要因素。在我国仅HBV慢性感染者就有1.25亿，HCV感染者也高达3800万。每年有大量的肝硬化病人死于包括原发性肝细胞癌在内的多种并发症。病毒感染者通常会经历病毒性肝炎——肝硬化——肝癌的典型病程，然而这一病程的确切机制并不清楚。病毒感染人体后与宿主细胞发生复杂的相互作用，其中涉及宿主细胞的多种基因、多种信号途径。分析感染后病变肝脏细胞基因的表达变化，不仅可以从一定程度上理解病毒致病的分子生物学，而且也为揭示病毒性肝硬化、原发性肝细胞癌的发病机理提供依据。同时这些差异表达的基因则可能成为新的肝病诊断标志或治疗靶点。
本研究选取正常肝脏组织和肝硬化组织(乙型肝炎病毒表面抗原阳性)、原发性肝细胞癌及癌旁组织(丙型肝炎病毒抗体阳性、核酸阳性)为研究对象，综合多个研究者对代表性差异分析(representational difference analysis，RDA)技术的改进方案分别进行了两组研究标本正向和反向的消减杂交，两轮杂交后的产物被克隆到pGEM-T载体中。挑取部分阳性克隆制备杂交膜，分别以同位素标记来源于各研究标本的扩增子以及第二轮杂交产物为探针，进行高通量的斑点杂交，获得多个差异表达的基因：(1)在肝硬化组织中上调的基因包括谷氨酸转氨酶(glutaminase 1)、醛糖还原酶(aldo-keto reductase family 1 B10，AKR1B10)、钙结合的酪氨酸磷酸化调节蛋白(calcium-binding tyrosine
博士学位论文摘要
phosphorylation一regulated protein，CABYR)、雌激素应答B盒蛋白(Estrogen
responsive B box protein，EBBP)等基因以及两个对应于新基因的EST(expressed
sequenee tag)片段等。下调表达的基因包括CD27结合蛋白(CD27 binding
protein珻D27BP)和烟酞胺甲基转移酶(nieotinamide
N一methyltransferase，NNMT)等。(2)在肝癌组织中上调表达的基因有微管蛋白
(gamma一tubulin)，丝氨酸脱氢酶(serine dehydratase，SDS)，P450色素家
族蛋白(eytoehrome P450，fami ly 11，subfamilyB，PolypeptideZ，CYPXIBZ)。
下调表达的基因包括与外毒素酶解代谢以及血浆蛋白的合成的相关基因以及肿瘤
相关基因丝氨酸蛋白酶11(protease，serine 11，HTRA serine protease，pRSSll)、
原肌球蛋白一1(Tropomyosin一l，TM一l)以及两个对应于新基因的EST片段等。选择
部分基因进一步用半定量RT一PCR的方法鉴定其在相应类型标本中的表达。CABYR
基因在正常肝脏中不表达，而在所有的13份肝硬化组织中都激活表达，同时钙结
合的酪氨酸磷酸化调节蛋白基因在7/9的肝癌组织中上调表达，表明该基因于转
录水平上在肝病发生中发挥重要作用。目前尚未有这一基因参与包括肝病在内的
人类疾病的相关研究。PRSS 11、TM一1基因在9份肝癌组织中呈现了不同的表达模
式。
通过本研究，可以得出如下结论:(1)利用RDA技术分离获得多个参与病毒
性肝硬化、肝癌发生的基因，其中包括一些新基因，EBBP、CABYR以及PRSSll基
因参与肝病的发生为我们首次报道。CABYR基因及其表达产物在肝癌发生中的作用
有待进一步研究。(2)结合RDA和高通量的再筛选可以有效分析细胞在病理状态下
差异表达的基因，是了解宿主和病毒相互作用的有效方法。
Hepatitis B/C virus (HBV/HCV) are the major etiologic agents of cirrhosis and hepatocellular carcinoma (HCC). In China, HBV chronically infects about 125 million, and HCV infects about 38 million people. Many HBV/HCV carriers usually develop chronic hepatitis(CH), finally complicated with HCC in a liver with cirrhosis or advanced stage CH. Because of interaction between virus and host, dynamic and closely related changes of gene expression are suspected to be involved in the pathogenesis of cirrhosis and hepatocarcinogenesis.
To gain insight into virus-host interrelationships and molecular portraits of cirrhosis and HCC, we used a modified complementary DNA representational difference analysis (cDNA-RDA) protocol to compare gene expression between normal liver and HBV cirrhosis, HCV-associated HCC and non-tumorous adjacent tissue. Different products (DP2) were cloned into pGEM-T vector. Positive clones were rescreened for differential expression by dot-blot hybridization with probes from amplicons and DP2. Selected genes were sequenced. Differentially expressed genes included: (1) In cirrhosis glutaminase 1, aldo-keto reductase family 1 B10 (AKR1B10) ,
calcium-binding tyrosine phosphorylation regulated protein (CABYR), estrogen responsive B box protein (EBBP) and two expressed sequence tags (EST) homologous with novel genes were up-regulated. CD27 binding proteinand nicotinamide N-methyltransferase (NNMT) were down-regulated. (2) In HCC gamma -tubulin, serine dehydratase(SDS), aldosterone synthase(CYPXI2B) and two new genes were up-regulated. The genes associated with xenobiotic catabolism and plasma protein synthesis and tumor associated genes PRSS11.TM-1 were down-regulated. Several differentially expressed genes were confirmed by semi-quantitative RT-PCR in the relevant tissues. CABYR gene were not expressed in normal liver, but expressed in cirrhosis tissue and overexpressed in 7/9 HCC tissues. This result indicates that CABYR gene plays an important role in pathogenesis of liver disease on transcription level. This is the first study that CABYR gene is involved in human disease. PRSS11 and TM-1 show differential expression pattern in 9 HCC tis
sues.
Conclusions: (1) Subtractive EST libraries of cirrhosis and HCC were constructed using modified cDNA-RDA. Novel observations of differential gene expression in liver disease were made. Further studies of CABYR gene and its products are warrented. (2) Combination of RDA and high-through rescreening can isolate differentially expressed genes involved in human disease successfully and obtain more insight into virus-host interaction.
本研究选取正常肝脏组织和肝硬化组织(乙型肝炎病毒表面抗原阳性)、原发性肝细胞癌及癌旁组织(丙型肝炎病毒抗体阳性、核酸阳性)为研究对象，综合多个研究者对代表性差异分析(representational difference analysis，RDA)技术的改进方案分别进行了两组研究标本正向和反向的消减杂交，两轮杂交后的产物被克隆到pGEM-T载体中。挑取部分阳性克隆制备杂交膜，分别以同位素标记来源于各研究标本的扩增子以及第二轮杂交产物为探针，进行高通量的斑点杂交，获得多个差异表达的基因：(1)在肝硬化组织中上调的基因包括谷氨酸转氨酶(glutaminase 1)、醛糖还原酶(aldo-keto reductase family 1 B10，AKR1B10)、钙结合的酪氨酸磷酸化调节蛋白(calcium-binding tyrosine
博士学位论文摘要
phosphorylation一regulated protein，CABYR)、雌激素应答B盒蛋白(Estrogen
responsive B box protein，EBBP)等基因以及两个对应于新基因的EST(expressed
sequenee tag)片段等。下调表达的基因包括CD27结合蛋白(CD27 binding
protein
N一methyltransferase，NNMT)等。(2)在肝癌组织中上调表达的基因有微管蛋白
(gamma一tubulin)，丝氨酸脱氢酶(serine dehydratase，SDS)，P450色素家
族蛋白(eytoehrome P450，fami ly 11，subfamilyB，PolypeptideZ，CYPXIBZ)。
下调表达的基因包括与外毒素酶解代谢以及血浆蛋白的合成的相关基因以及肿瘤
相关基因丝氨酸蛋白酶11(protease，serine 11，HTRA serine protease，pRSSll)、
原肌球蛋白一1(Tropomyosin一l，TM一l)以及两个对应于新基因的EST片段等。选择
部分基因进一步用半定量RT一PCR的方法鉴定其在相应类型标本中的表达。CABYR
基因在正常肝脏中不表达，而在所有的13份肝硬化组织中都激活表达，同时钙结
合的酪氨酸磷酸化调节蛋白基因在7/9的肝癌组织中上调表达，表明该基因于转
录水平上在肝病发生中发挥重要作用。目前尚未有这一基因参与包括肝病在内的
人类疾病的相关研究。PRSS 11、TM一1基因在9份肝癌组织中呈现了不同的表达模
式。
通过本研究，可以得出如下结论:(1)利用RDA技术分离获得多个参与病毒
性肝硬化、肝癌发生的基因，其中包括一些新基因，EBBP、CABYR以及PRSSll基
因参与肝病的发生为我们首次报道。CABYR基因及其表达产物在肝癌发生中的作用
有待进一步研究。(2)结合RDA和高通量的再筛选可以有效分析细胞在病理状态下
差异表达的基因，是了解宿主和病毒相互作用的有效方法。
Hepatitis B/C virus (HBV/HCV) are the major etiologic agents of cirrhosis and hepatocellular carcinoma (HCC). In China, HBV chronically infects about 125 million, and HCV infects about 38 million people. Many HBV/HCV carriers usually develop chronic hepatitis(CH), finally complicated with HCC in a liver with cirrhosis or advanced stage CH. Because of interaction between virus and host, dynamic and closely related changes of gene expression are suspected to be involved in the pathogenesis of cirrhosis and hepatocarcinogenesis.
To gain insight into virus-host interrelationships and molecular portraits of cirrhosis and HCC, we used a modified complementary DNA representational difference analysis (cDNA-RDA) protocol to compare gene expression between normal liver and HBV cirrhosis, HCV-associated HCC and non-tumorous adjacent tissue. Different products (DP2) were cloned into pGEM-T vector. Positive clones were rescreened for differential expression by dot-blot hybridization with probes from amplicons and DP2. Selected genes were sequenced. Differentially expressed genes included: (1) In cirrhosis glutaminase 1, aldo-keto reductase family 1 B10 (AKR1B10) ,
calcium-binding tyrosine phosphorylation regulated protein (CABYR), estrogen responsive B box protein (EBBP) and two expressed sequence tags (EST) homologous with novel genes were up-regulated. CD27 binding protein
sues.
Conclusions: (1) Subtractive EST libraries of cirrhosis and HCC were constructed using modified cDNA-RDA. Novel observations of differential gene expression in liver disease were made. Further studies of CABYR gene and its products are warrented. (2) Combination of RDA and high-through rescreening can isolate differentially expressed genes involved in human disease successfully and obtain more insight into virus-host interaction.
引文
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2 Hildt E, Hofschneider PH. The PreS2 activators of the hepatitis B virus: activators of tumour promoter pathways. Recent Results Cancer Res. 1998,154:315-29
3 Podevin P, Saible A, Gajardo R, et al. Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner. Hepatl. 2001, 33(6):1503-1511
4 Sakamuro D, Furukawa T, Takegami T, et al. Hepatitis C virus non structural protein NS3 transforms NIH 3T3 Cells.J Virol, 1995,69(6):3893-3896
5 Ray RB, Ray R. Hepatitis C virus core protein: intriguing properties and functional relevance, FEMS Microbiol lett, 202:149-156
6 Pastorian K, Hawel L 3rd, Byus CV. Optimization of cDNA representational difference analysis for the indentification of differetially expressed mRNAs. Anal Biochem, 2000, 283(1):89-98.
7 Welford SM, Gregg J,Chen E, et al. Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray, hybridization. Nucl Acids Res, 1998,26(12):3059-3065.
1 Pastorian K, Hawel L 3rd, Byus CV. Optimization of cDNA representational difference analysis for the indentification of differetially expressed
mRNAs. Anal Biochem, 2000, 283(1):89-98.
2 Geng M, Wallrapp C, Muller-Pillasch F, et al. Isolation of differentially expressed genes by combining representational difference analysis(RDA) and cDNA library arrays. Biotechniques, 1998,25(3):434-438.
3 Welford SM, Gregg J, Chen E, et al. Detection ofdifferentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucl Acids Res, 1998,26(12):3059-3065.
4 Wang SM, Fears SC, zhang L, et al. Screening poly(dA/dT)- cDNAs for gene identification. Proc Natl Acad Sci USA, 2000, 97(8):4162-4167.
5 Nam DK, Lee S, Zhou G, et al. Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) primeing during reverse transcription. Proc Natl Acad Sci USA, 2002, 99(9):6152-6156.
6 Scuric Z, Stain SC, Anderson WF, et al. New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma. Hepatol, 1998,27(4): 943-950.
7 Liu HL, Golder-Novoselsky E, Seto MH, et al. The novel estrogen-responsive B- box protein(EBBP) gene is tamoxifen regulated in cells expressing an estrogen receptor DNA-binding domain mutant. Mol Endocri, 1998,12(1):1733-48.
8 Beer HD, Munding C, Duboist N, et al. The estrogen-responsive B box protein : a novel regulatior of kerationocyte differentiation. J Bio Chem, 2002, 277(23):20740-20749.
9 Naaby-Hansen S, Mandal A, Wolkowicz MJ, et al. CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation. Dev Biol, 2002, 242(2):236-254.
10 Sen B, Mandal A, Wolkowicz MJ, et al. Splicing in murine CABYR and its genomic strcture. Gene, 2003, 310:67-78.
11 Shackel NA, McGuinness PH, Abbott CA, et al. Insights into the pathobiology of hepatitis C virus-associated cirrhosis. Amer J Pathol, 2002, 160(2):641-654.
12 Kim MY, Park E, Park JH, et al. Expression profile of nine novel genes differentially expressed in hepatitis B virus-associated hepatocellular carcinomas. Oncogene, 2001,20:4568-4575
1 Cohen J. The scientific challenge of hepatitis C. Science, 1999, 285:26-30
2 Delhem N, Sabile A, Gajardo R, et al. Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein. Oncogene. 2001 ,20(41):5836-45.
3 Hayashi J, Aoki H, Arakawa Y, et al. Hepatitis C virus core protein activates the MAPK/ERK cascade synergistically with tumor promoter TPA, but not with epidermal growth factor or transforming growth factor alpha. Hepatology. 2000,32(5):958-61.
4 Podevin P, Saible A, Gajardo R,et al. Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner. Hepatl. 2001,33(6):1503-1511
5 sakamuro D, Furukawa T, Takegami T, et al. Hepatitis C virus non structural protein NS3 transforms NIH 3T3 cells. J Virol, 1995,69(6):3893-3896
6 Ray RB, Ray R. Hepatitis C.virus core protein: intriguing properties and functional relevance, FEMS Microbiol lett, 202:149-156
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1 Naaby-Hansen S, Mandal A, Wolkowicz MJ, et al. CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath
protein involved in capacitation. Developmental Biology, 2002,242:236-254
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3 Takahashi M, Saito H, Atsukawa K, et al. Bcl-2 prevents doxorubicin-induced apoptosis of human liver cancer cells. Hepatol Res, 2003,25(2):192-201
4 Shirota Y, KanekoS, HondaM, et al. Identification of differentially expressed genes in hepatocellular carcinomas with cDNA microarrays. Hepatol, 2001,33(4):832-840
5 Tannapfel A, Anhalt K, Hausermann P, et al. Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays. J Pathol, 2003,20I(2):238-49
6 F．奥斯伯，R．布伦特，R．E．金斯顿等著，颜子颖，王海林译．精编分子生物学实验指南．科学出版社，1998年，第一版
7 李永明，赵玉琪等著，李电东，陈巍等译．实用分子生物学方法手册．科学出版社，1998：76-77
2 Hildt E, Hofschneider PH. The PreS2 activators of the hepatitis B virus: activators of tumour promoter pathways. Recent Results Cancer Res. 1998,154:315-29
3 Podevin P, Saible A, Gajardo R, et al. Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner. Hepatl. 2001, 33(6):1503-1511
4 Sakamuro D, Furukawa T, Takegami T, et al. Hepatitis C virus non structural protein NS3 transforms NIH 3T3 Cells.J Virol, 1995,69(6):3893-3896
5 Ray RB, Ray R. Hepatitis C virus core protein: intriguing properties and functional relevance, FEMS Microbiol lett, 202:149-156
6 Pastorian K, Hawel L 3rd, Byus CV. Optimization of cDNA representational difference analysis for the indentification of differetially expressed mRNAs. Anal Biochem, 2000, 283(1):89-98.
7 Welford SM, Gregg J,Chen E, et al. Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray, hybridization. Nucl Acids Res, 1998,26(12):3059-3065.
1 Pastorian K, Hawel L 3rd, Byus CV. Optimization of cDNA representational difference analysis for the indentification of differetially expressed
mRNAs. Anal Biochem, 2000, 283(1):89-98.
2 Geng M, Wallrapp C, Muller-Pillasch F, et al. Isolation of differentially expressed genes by combining representational difference analysis(RDA) and cDNA library arrays. Biotechniques, 1998,25(3):434-438.
3 Welford SM, Gregg J, Chen E, et al. Detection ofdifferentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucl Acids Res, 1998,26(12):3059-3065.
4 Wang SM, Fears SC, zhang L, et al. Screening poly(dA/dT)- cDNAs for gene identification. Proc Natl Acad Sci USA, 2000, 97(8):4162-4167.
5 Nam DK, Lee S, Zhou G, et al. Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) primeing during reverse transcription. Proc Natl Acad Sci USA, 2002, 99(9):6152-6156.
6 Scuric Z, Stain SC, Anderson WF, et al. New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma. Hepatol, 1998,27(4): 943-950.
7 Liu HL, Golder-Novoselsky E, Seto MH, et al. The novel estrogen-responsive B- box protein(EBBP) gene is tamoxifen regulated in cells expressing an estrogen receptor DNA-binding domain mutant. Mol Endocri, 1998,12(1):1733-48.
8 Beer HD, Munding C, Duboist N, et al. The estrogen-responsive B box protein : a novel regulatior of kerationocyte differentiation. J Bio Chem, 2002, 277(23):20740-20749.
9 Naaby-Hansen S, Mandal A, Wolkowicz MJ, et al. CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation. Dev Biol, 2002, 242(2):236-254.
10 Sen B, Mandal A, Wolkowicz MJ, et al. Splicing in murine CABYR and its genomic strcture. Gene, 2003, 310:67-78.
11 Shackel NA, McGuinness PH, Abbott CA, et al. Insights into the pathobiology of hepatitis C virus-associated cirrhosis. Amer J Pathol, 2002, 160(2):641-654.
12 Kim MY, Park E, Park JH, et al. Expression profile of nine novel genes differentially expressed in hepatitis B virus-associated hepatocellular carcinomas. Oncogene, 2001,20:4568-4575
1 Cohen J. The scientific challenge of hepatitis C. Science, 1999, 285:26-30
2 Delhem N, Sabile A, Gajardo R, et al. Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein. Oncogene. 2001 ,20(41):5836-45.
3 Hayashi J, Aoki H, Arakawa Y, et al. Hepatitis C virus core protein activates the MAPK/ERK cascade synergistically with tumor promoter TPA, but not with epidermal growth factor or transforming growth factor alpha. Hepatology. 2000,32(5):958-61.
4 Podevin P, Saible A, Gajardo R,et al. Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner. Hepatl. 2001,33(6):1503-1511
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