DcR3基因扩增和表达与肝癌细胞凋亡、化疗抗性关系的实验研究
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摘要
Decory receptor 3(DcR3)是1998年由Pitti RM等首先发现的一种陷阱分子，属于肿瘤坏死因子受体超家族的成员。DcR3基因定位于染色体20q13.3上，该基因编码一种无明显跨膜序列的分泌型蛋白，能特异性地结合FasL，阻断由Fas／FasL系统介导的细胞凋亡。有作者在研究中发现人类的多种恶性肿瘤(如肺，胃肠道组织、淋巴细胞、神经组织等的恶性肿瘤)中均存在DcR3基因的过度表达和扩增，而且这种过度表达仅发生于肿瘤细胞，肿瘤周围的正常组织则检测不到该基因的表达。这些研究结果陆续发表在《Nature》和《Science》等刊物上。随后在DcR3基因的功能研究中发现：DcR3基因的过度表达与肿瘤的免疫逃逸及抵抗机体的细胞凋亡机制有关，在肿瘤的发生和发展中起重要作用。许多肿瘤治疗研究的突破与改变肿瘤细胞凋亡机制密切相关。诱导凋亡治疗将成为人类征服肿瘤的有力手段。纵观国外的研究结果，DcR3在恶性肿瘤中的过度表达和扩增是一个非常值得我们关注的课题。
DcR3在肿瘤免疫逃逸中的重要作用提示我们，如果能从蛋白水平甚至基因水平阻断肿瘤组织DcR3的表达，将积极推动DcR3阳性肿瘤的治疗，甚至阻断恶性肿瘤的形成或复发。由于DcR3表达分泌型蛋白，因此，血清中的DcR3蛋白是一个早期筛查DcR3阳性表达肿瘤的很有诊断价值的分子Marker。我们在这方面已进行了初步研究，在肝癌等许多恶性肿瘤病人的血清中检测到DcR3蛋白的明显升高，部分阳性病人可达正常对照人群的20倍。然而，DcR3基因在原发性肝癌中的表达以及扩增情况，该基因表达与肝癌细胞凋亡的关系、在肝癌形成中的作用等，国内外尚未见研究报道。肝癌是人类常见的恶性肿瘤，是危害人类健康的重大疾病。通过近半个世纪多学科、多中心的共同努力，人们在肝癌的病因、诊断及治疗等多方面均取得了突破性的进展。但肝癌仍在肆虐着人类的生命，对肝癌我们还远远未达到基本控制的地步．由于DcR3基因表达与肿瘤细胞抵抗机体细胞凋亡机制之间的密切关系，研究DcR3基因在肝癌细胞抵抗凋亡中的作用是一个很有价值的课题。
浙江大学博士学位论文
根据国内外的研究动态，本课题利用先进的分子生物学、免疫组织化学以及细胞学
研究技术，研究肝癌中IkR3基因的表达和扩增情况，分析基因扩增在过度表达中的作
用，并进一步观察该基因表达在肝癌细胞凋亡和增殖发生中的作用；此外，我们还对DcR3
基因表达在肝癌细胞化疗抗性中的作用作出了初步探讨。研究结果为弄清肝癌抵抗机体
的细胞凋亡中的作用机制奠定一定的实验和理论基础，有望为肝癌的诱导凋亡治疗开辟
一个全新的研究领域。
第一部分：原发 癌中DcR3基因的表达和扩憎及其意义
目的：研究DCR3基因在原发性肝癌中的表达和扩增情况，探讨该基因在肝癌中过度表
达的原因，比较具有不同临床及病理学因素的肝癌其D。R3基因表达和扩增的差
异。
方法：选择48例经手术和病理学检查证实的原发性肝癌病人的组织，运用r－PCR检测
癌组织及癌旁肝组织中的DcR3基因mRNA表达，荧光定量PCR检测肝癌组织中该
基因的扩增情况，SABC免疫组织化学染色原位检测肝癌组织中D。R3蛋白的表达。
统计分析肝癌中DcR3基因表达与该基因扩增的相关性，研究DcR3基因在肝癌中
过度表达的原因，比较不同临床及病理学类型肝癌中该基因表达及扩增的差异。
结果：48例肝癌组织中DCR3基因CRNA阳性表达29例，阳性表达率为60．4％（29／48）；
而对应的癌旁组织中未见阳性表达。该基因的mRNA表达与肿瘤大小、临床分期
及肿瘤是否有浸润和转移有关（P＜0．05），但与肿瘤有无包膜、癌栓及肿瘤的结节
数目无明显相关性（P＞0．05）。DcR3 基因 mRNA阳性表达组的扩增倍数为
l．53士0．82，阴性表达组的扩增倍数为0．75士0 33，两者的差异有显著性
（P＜0．of）。
48例病人的DeR3基因扩增倍数为0．18～3．86，平均值为1．22 士0．77，扩
增倍数超过2的病人共7例，占14．6％（7／48）。以扩增倍数o、刀将48例病
人分成两组，比较不同扩增倍数肝癌病人该基因毗肌表达的差异，结果发现其
差异有显著性（P＜0．of）；具有不同临床及病理学特征的肝癌，其 DCR3基因的扩
增倍数无明显差异（P＞0．05）。在对RT－PCR扩增产物进行测序分析的研究结果中，
发现有3个位点存在点突变。
肝癌组织中DcR3蛋白表达的免疫纽化检测结果发现：肝癌组织中存在D。R3
蛋白的阳性表达，其阳性信号位于肝癌细胞的胞浆中，周围正常的肝组织未见阳
DcR3 (also known as TR6 and M68), a new member of the TNF receptor (TNFR) family, has been identified by Pitti et al in 1998. It maps to chromosome position 20ql3.3. DcR3 is a soluble decoy receptor for FasL,which lacks a transmembrance domain and is secreted into the extracellular space. DcR3 binds to the FasL and LIGHT, thereby neutralizing their proapoptotic action. It has been demonstrated that DcR3 frequently is overexpressed in malignant tumors arising from lung, gastrointestinal tract and et al. The DcR3 overexpression exists only in tumor tissues. Moreover, Pitti et al detected a high percentage of DcR3 gene amplifications in colon and lung carcinoma. Thus it has been postulated that Fas/FasL interactions might limit cancer growth and that cells expressing high levels of DcR3 are more likely to escape elimination via the Fas/FasL pathway. Apoptosis is known to be very important in tumor biogressiveness. A lot of strages for treatment of tumor are based on involving the mechanism of apoptosis. To trea
t tumor via inducing apoptosis is more and more improtant, and is going to be a powerful weapon which is used to conquer tumor by people.
The function of DcR3 in immune evasion remind us, if the DcR3 expression can be suppressed in the level of gene or protein, then the treatment of tumor will be improved, moreover we can stop the information and recurrence of tumor. DcR3 is a soluble protein, thus detecting DcR3 protein level in blood may be a valuable diagnostic method for the tumor with positive DcR3 protein expression. We have already done one pilot investion for serum level of DcR3 protein in many kind of tumors. We find that the level of DcR3 protein in peripheral blood is significantly higher in patients with HCC than in healthy people. Compare with the healthy people, some patients with HCC have a 20-fold DcR3 protein level. But until
now, there are no research on the DcR3 expression and genomic amplification in HCC, on the relationship between the DcR3 expression and apoptosis of HCC.
In this study, the mRNA expression and genomic amplification of DcR3 in HCC were investigated , the values of DcR3 overexpression in suppressing FasL-induced apoptosis and chemotaxis was analyzed. This study is aimed at investigating the antiapoptosis mechanism of HCC. The result of this study will be a fundamental in treatment of HCC via the way inducing apoptosis.
Part I: The overexpression and genomic amplification of DcR3 and its significance Objective:To study the mRNA expression and genomic amplification of DcR3 in HCC, to
analyze the mechanism of DcR3 overexpression.
Methods: 48 cases of HCC, which has been proved by operation and pathology, is studied. Detecting the DcR3 mRNA in the cancer tissues and the normal tissues adjacent to the tumor with RT-PCR, quantifing the DcR3 gene amplification in the cancer tissues by quantitative genomic PCR, examining DcRJ protein expression in the tumor tissues of HCC by SABC immunohistochemistry (IHC).To analyze the relationship between the DcR3 mRNA expression and genomic amplification of HCC with statistic methods, to compare the difference of DcR3 expression and amplification in the HCC with different clinical and pathological feature.
Results: DcR3 mRNA was detected in 29 cases out of total 48 case HCC, the ratio of positive expression is 60.4%(29/48). No positive mRNA expression was detected in 48 normal tissues adjacent to the tumor. DcR3 mRNA expression in HCC show a relationship with the size of mass, the clinical stage and the metastasis of the tumor(P<0.05)J but no relationship with the membrane of the tumor, cancer embolus, and the number of mass. The DcR3 gene amplification in 48 cases of HCC ranges from 0.18 to 3.86-fold. 7 of 48 cases HCC show significantly higher DcR3 gene amplification than the normal control tissues. Compare the DcR3 mRNA expression between the patients with <1.0 and >1.0 gene amplification fold, the difference show a statistic significance(P<0.01). The DcR3 gene amplification fold of the patients with positive mRNA expressio
DcR3在肿瘤免疫逃逸中的重要作用提示我们，如果能从蛋白水平甚至基因水平阻断肿瘤组织DcR3的表达，将积极推动DcR3阳性肿瘤的治疗，甚至阻断恶性肿瘤的形成或复发。由于DcR3表达分泌型蛋白，因此，血清中的DcR3蛋白是一个早期筛查DcR3阳性表达肿瘤的很有诊断价值的分子Marker。我们在这方面已进行了初步研究，在肝癌等许多恶性肿瘤病人的血清中检测到DcR3蛋白的明显升高，部分阳性病人可达正常对照人群的20倍。然而，DcR3基因在原发性肝癌中的表达以及扩增情况，该基因表达与肝癌细胞凋亡的关系、在肝癌形成中的作用等，国内外尚未见研究报道。肝癌是人类常见的恶性肿瘤，是危害人类健康的重大疾病。通过近半个世纪多学科、多中心的共同努力，人们在肝癌的病因、诊断及治疗等多方面均取得了突破性的进展。但肝癌仍在肆虐着人类的生命，对肝癌我们还远远未达到基本控制的地步．由于DcR3基因表达与肿瘤细胞抵抗机体细胞凋亡机制之间的密切关系，研究DcR3基因在肝癌细胞抵抗凋亡中的作用是一个很有价值的课题。
浙江大学博士学位论文
根据国内外的研究动态，本课题利用先进的分子生物学、免疫组织化学以及细胞学
研究技术，研究肝癌中IkR3基因的表达和扩增情况，分析基因扩增在过度表达中的作
用，并进一步观察该基因表达在肝癌细胞凋亡和增殖发生中的作用；此外，我们还对DcR3
基因表达在肝癌细胞化疗抗性中的作用作出了初步探讨。研究结果为弄清肝癌抵抗机体
的细胞凋亡中的作用机制奠定一定的实验和理论基础，有望为肝癌的诱导凋亡治疗开辟
一个全新的研究领域。
第一部分：原发 癌中DcR3基因的表达和扩憎及其意义
目的：研究DCR3基因在原发性肝癌中的表达和扩增情况，探讨该基因在肝癌中过度表
达的原因，比较具有不同临床及病理学因素的肝癌其D。R3基因表达和扩增的差
异。
方法：选择48例经手术和病理学检查证实的原发性肝癌病人的组织，运用r－PCR检测
癌组织及癌旁肝组织中的DcR3基因mRNA表达，荧光定量PCR检测肝癌组织中该
基因的扩增情况，SABC免疫组织化学染色原位检测肝癌组织中D。R3蛋白的表达。
统计分析肝癌中DcR3基因表达与该基因扩增的相关性，研究DcR3基因在肝癌中
过度表达的原因，比较不同临床及病理学类型肝癌中该基因表达及扩增的差异。
结果：48例肝癌组织中DCR3基因CRNA阳性表达29例，阳性表达率为60．4％（29／48）；
而对应的癌旁组织中未见阳性表达。该基因的mRNA表达与肿瘤大小、临床分期
及肿瘤是否有浸润和转移有关（P＜0．05），但与肿瘤有无包膜、癌栓及肿瘤的结节
数目无明显相关性（P＞0．05）。DcR3 基因 mRNA阳性表达组的扩增倍数为
l．53士0．82，阴性表达组的扩增倍数为0．75士0 33，两者的差异有显著性
（P＜0．of）。
48例病人的DeR3基因扩增倍数为0．18～3．86，平均值为1．22 士0．77，扩
增倍数超过2的病人共7例，占14．6％（7／48）。以扩增倍数o、刀将48例病
人分成两组，比较不同扩增倍数肝癌病人该基因毗肌表达的差异，结果发现其
差异有显著性（P＜0．of）；具有不同临床及病理学特征的肝癌，其 DCR3基因的扩
增倍数无明显差异（P＞0．05）。在对RT－PCR扩增产物进行测序分析的研究结果中，
发现有3个位点存在点突变。
肝癌组织中DcR3蛋白表达的免疫纽化检测结果发现：肝癌组织中存在D。R3
蛋白的阳性表达，其阳性信号位于肝癌细胞的胞浆中，周围正常的肝组织未见阳
DcR3 (also known as TR6 and M68), a new member of the TNF receptor (TNFR) family, has been identified by Pitti et al in 1998. It maps to chromosome position 20ql3.3. DcR3 is a soluble decoy receptor for FasL,which lacks a transmembrance domain and is secreted into the extracellular space. DcR3 binds to the FasL and LIGHT, thereby neutralizing their proapoptotic action. It has been demonstrated that DcR3 frequently is overexpressed in malignant tumors arising from lung, gastrointestinal tract and et al. The DcR3 overexpression exists only in tumor tissues. Moreover, Pitti et al detected a high percentage of DcR3 gene amplifications in colon and lung carcinoma. Thus it has been postulated that Fas/FasL interactions might limit cancer growth and that cells expressing high levels of DcR3 are more likely to escape elimination via the Fas/FasL pathway. Apoptosis is known to be very important in tumor biogressiveness. A lot of strages for treatment of tumor are based on involving the mechanism of apoptosis. To trea
t tumor via inducing apoptosis is more and more improtant, and is going to be a powerful weapon which is used to conquer tumor by people.
The function of DcR3 in immune evasion remind us, if the DcR3 expression can be suppressed in the level of gene or protein, then the treatment of tumor will be improved, moreover we can stop the information and recurrence of tumor. DcR3 is a soluble protein, thus detecting DcR3 protein level in blood may be a valuable diagnostic method for the tumor with positive DcR3 protein expression. We have already done one pilot investion for serum level of DcR3 protein in many kind of tumors. We find that the level of DcR3 protein in peripheral blood is significantly higher in patients with HCC than in healthy people. Compare with the healthy people, some patients with HCC have a 20-fold DcR3 protein level. But until
now, there are no research on the DcR3 expression and genomic amplification in HCC, on the relationship between the DcR3 expression and apoptosis of HCC.
In this study, the mRNA expression and genomic amplification of DcR3 in HCC were investigated , the values of DcR3 overexpression in suppressing FasL-induced apoptosis and chemotaxis was analyzed. This study is aimed at investigating the antiapoptosis mechanism of HCC. The result of this study will be a fundamental in treatment of HCC via the way inducing apoptosis.
Part I: The overexpression and genomic amplification of DcR3 and its significance Objective:To study the mRNA expression and genomic amplification of DcR3 in HCC, to
analyze the mechanism of DcR3 overexpression.
Methods: 48 cases of HCC, which has been proved by operation and pathology, is studied. Detecting the DcR3 mRNA in the cancer tissues and the normal tissues adjacent to the tumor with RT-PCR, quantifing the DcR3 gene amplification in the cancer tissues by quantitative genomic PCR, examining DcRJ protein expression in the tumor tissues of HCC by SABC immunohistochemistry (IHC).To analyze the relationship between the DcR3 mRNA expression and genomic amplification of HCC with statistic methods, to compare the difference of DcR3 expression and amplification in the HCC with different clinical and pathological feature.
Results: DcR3 mRNA was detected in 29 cases out of total 48 case HCC, the ratio of positive expression is 60.4%(29/48). No positive mRNA expression was detected in 48 normal tissues adjacent to the tumor. DcR3 mRNA expression in HCC show a relationship with the size of mass, the clinical stage and the metastasis of the tumor(P<0.05)J but no relationship with the membrane of the tumor, cancer embolus, and the number of mass. The DcR3 gene amplification in 48 cases of HCC ranges from 0.18 to 3.86-fold. 7 of 48 cases HCC show significantly higher DcR3 gene amplification than the normal control tissues. Compare the DcR3 mRNA expression between the patients with <1.0 and >1.0 gene amplification fold, the difference show a statistic significance(P<0.01). The DcR3 gene amplification fold of the patients with positive mRNA expressio
引文
[1] Oldham BK. Biotherapy: the fourth modality of cancer treatment. J Cell Physiol, 1986;4:91
[2] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science, 1995,267:1456
[3] Carson DN, Ribero JM. Apoptosis and disease. Lancet, 1993;341:1251
[4] Marx J. Cell death studies yield cancer clues. Science, 1995,259:760
[5] Eastman A, Grant S, Lock R, et al. Cell death in cancer and development. Cancer Res, 1994;54:2812
[6] Kerr JFR, Winterford CM, Haarmon BV, et al. Apoptosis: its significance in cancer therapy. Cancer, 1994;73:2113
[7] Harrison DJ. Life and death in the genesis of the tumor cell. Toxicology Letters, 1998;102:115-119
[8] Pitti-RM, Marsters-SA, Lawrence-DA, et.al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature. 1998 ; 396(6712) : 699-703
[9] Schwendel-A, Richard-F, Langreck-H, et al. Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chromosomes 4q and 21q. Br J Cancer.1998; 78:806-811
[10] Sakakura-C, Mori-T, Sakabe-T, et al. Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer. 1999;24:299-305
[11] Muleris-M, Almeida-A, Gerbault Seureau-M, et al. Identification of amplified DNA sequences in breast cancer and their organization within homogeneously staining regions. Genes Chromosomes Cancer. 1995;14(3) :155-63
[12] Altura-RA, Maris-JM, Li-H, et al. Novel regions of chromosomal loss in familial neuroblastoma by comparative genomic hybridization. Genes Chromosomes Cancer. 1997; 19:176-184
[13] Shinomiya-T, Mori-T, Ariyama-Y, et al. Comparative genomic hybridization of squamous cell carcinoma of the esophagus: the possible involvement of the DPI gene in the 13q34 amplicon. Genes Chromosomes Cancer. 1999;24:337-344
[14] Bai-C, Connolly-B, Metzker-ML, et al. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc-Natl-Acad-Sci-U-S-A. 2000 ; 97(3) : 1230-5
[15] Roth-W, Isenmann-S, Nakamura-M, et al. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Research. 2001 ;61:2759-2765
[16] Yu-KY, Kwon-B, Ni-J, et al. A newly identified member of tumor necrosis factor receptor superfamily(TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem. 1999;274(20) :13733-6
[17] Ohshima K, Haraoka S, Sugihara M, et al. Amplification and expression of a decoy receptor for fas ligand (DcR3) in virus (EBV or HTLV-I) associated lymphomas. Cancer Lett 2000;160(1) :89-97
[18] Ibrahim SM, Ringel J, Schmidt C, et al. Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death. Pancreas 2001 Jul;23(1) :72-9
[19] Maeda T, Hao C, Tron VA. Ultraviolet Light (UV) Regulation of the TNF Family Decoy Receptors DcR2 and DcR3 in Human Keratinocytes. J Cutan Med Surg 2001;5(4) :294-8
[2] Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science, 1995,267:1456
[3] Carson DN, Ribero JM. Apoptosis and disease. Lancet, 1993;341:1251
[4] Marx J. Cell death studies yield cancer clues. Science, 1995,259:760
[5] Eastman A, Grant S, Lock R, et al. Cell death in cancer and development. Cancer Res, 1994;54:2812
[6] Kerr JFR, Winterford CM, Haarmon BV, et al. Apoptosis: its significance in cancer therapy. Cancer, 1994;73:2113
[7] Harrison DJ. Life and death in the genesis of the tumor cell. Toxicology Letters, 1998;102:115-119
[8] Pitti-RM, Marsters-SA, Lawrence-DA, et.al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature. 1998 ; 396(6712) : 699-703
[9] Schwendel-A, Richard-F, Langreck-H, et al. Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chromosomes 4q and 21q. Br J Cancer.1998; 78:806-811
[10] Sakakura-C, Mori-T, Sakabe-T, et al. Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer. 1999;24:299-305
[11] Muleris-M, Almeida-A, Gerbault Seureau-M, et al. Identification of amplified DNA sequences in breast cancer and their organization within homogeneously staining regions. Genes Chromosomes Cancer. 1995;14(3) :155-63
[12] Altura-RA, Maris-JM, Li-H, et al. Novel regions of chromosomal loss in familial neuroblastoma by comparative genomic hybridization. Genes Chromosomes Cancer. 1997; 19:176-184
[13] Shinomiya-T, Mori-T, Ariyama-Y, et al. Comparative genomic hybridization of squamous cell carcinoma of the esophagus: the possible involvement of the DPI gene in the 13q34 amplicon. Genes Chromosomes Cancer. 1999;24:337-344
[14] Bai-C, Connolly-B, Metzker-ML, et al. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc-Natl-Acad-Sci-U-S-A. 2000 ; 97(3) : 1230-5
[15] Roth-W, Isenmann-S, Nakamura-M, et al. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Research. 2001 ;61:2759-2765
[16] Yu-KY, Kwon-B, Ni-J, et al. A newly identified member of tumor necrosis factor receptor superfamily(TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem. 1999;274(20) :13733-6
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