摘要
研究背景
胆囊癌在胆道系统恶性肿瘤中位居首位,在整个消化系统的恶性肿瘤中排名第5位。胆囊癌恶性程度极高,早期无特异表现,所以很多病人在就诊时已为晚期。仅有20%-30%的胆囊癌患者可以得到根治性手术切除,而胆囊癌对于化疗和放疗均不敏感,所以从基因角度探索胆囊癌的治疗具有重要意义。
分化相关基因NDRG1是荷兰学者vanBelzen等1997年从结肠癌HT29-D4细胞株体外诱导分化过程中克隆到的一个新基因,该基因存在于人体多种组织,如肾脏、心、脑、骨骼肌、胎盘、肺组织、前列腺、小肠、大肠和卵巢等均有较高表达,而在外周血白细胞、免疫器官、肝脏等组织呈低表达,其表达的广泛性提示,该基因在多种组织细胞的分化及生长过程中可能起着重要作用。
Kurdistani等研究发现,该基因在多种肿瘤细胞株和肿瘤组织,如乳腺癌、前列腺癌和结直肠腺癌等肿瘤组织中均呈低表达。但也有研究认为,肿瘤组织NDRG1基因表达显著升高。Cnaugl的研究认为,肺癌、脑瘤、黑色素瘤、肝癌、前列腺癌、乳腺癌和肾癌等癌组织NDRG1蛋白表达较正常对照组织显著升高,其表达升高是由于这些癌组织缺氧引起的,NDRG1可作为一种重要的衡量组织缺氧的标志物。
但是,迄今尚无关于NDRG1基因在胆囊癌中表达的文献报道,对于该基因在胆囊癌中作用机制的研究则更是完全空白。因此,本研究拟应用各种分子和细胞生物学实验手段首次阐明NDRG1基因在胆囊癌中的表达状况,并进一步探讨其临床病理和预后意义以及相关分子机制。
本实验的基本思路是：为系统地探讨NRDG1在胆囊癌的表达情况,研究其与胆囊癌发生、发展的关系,了解各种细胞增殖相关蛋白对GBC-SD细胞NDRG1表达的影响及NDRG1基因表达在细胞增殖中的作用,本研究采用免疫组化法、Western Blot法等技术,检测了141例胆囊癌组织和癌旁正常粘膜中NDRG1的表达情况；采用免疫细胞化学染色、免疫印迹法(Western Blot)检测NDRG1在胆囊癌GBC-SD细胞中的表达情况；采用基因转染过表达策略,通过对下游基因和表型的改变(主要要包括细胞增殖、侵袭和凋亡三个方而)进行观察和分析NDRG1基因在胆囊癌细胞中表达情况及其与肿瘤生长、细胞增殖和细胞周期变化的关系。通过以上实验,试图阐明NDRG1在胆囊癌的表达规律；探讨NDRG1基因在肿瘤细胞诱导分化过程中的作用；研究GBC-SD细胞NDRG1基因表达与肿瘤生长、细胞增殖、细胞周期的变化等的关系；以进一步了解NDRG1基因在胆囊癌诱导分化中的作用机制,为胆囊癌诱导分化治疗提供新的理论依据。
第一章NDRG1编码蛋白在胆囊癌的表达、临床病理和预后意义
目的N-myc下游调节基因1(NDRG1),是N-myc下游调节基因家族成员之一,在多种应激反应和细胞生长调节情况下均可被诱导产生。最近已经有研究证实,NDRG1在多种不同类型的肿瘤中的作用机制与抑制肿瘤转移相关,并推测NDRG1可能是一个肿瘤抑制基因。到目前为止,尚未有报道关于NDRG1在胆囊癌的作用机制。因此,本研究的目的是调查NDRG1在GBC的表达情况及其对愈后预测的作用。
方法选取了141例胆囊癌患者的肿瘤组织标本,运用免疫组化方法分析NDRG1在其中的表达情况,NDRG1表达和临床病理因素、生存率的关系。
结果NDRG1在GBC表达的比例为63.8%；NDRG1在正常的胆囊壁上皮组织中没有表达,而在肿瘤侵袭前沿细胞则表达显著；在组织学分级高、病理分期和临床分期晚、淋巴转移阳性、静脉／淋巴受侵情况下,NDRG1表达明显；此外, Kaplan-Meier曲线提示,NDRG1过度表达和总体生存率、无病生存率方面有明显的负相关性(二者P值=0.02)。此外,多因素分析显示NDRG1表达(风险比：3.338；P=0.02),临床分期(风险比：3.128；P=0.03)均是影响无瘤生存率的独立危险因素。
结论通过本次研究,我们首次得出这些数据,NDRG1在GBC表达和临床病理的恶性程度相关,是一个独立的预示着患者无瘤生存率低的信息。我们的发现提示,在GBC中,NDRG1表达可能是一种新型的预测患者生存率的信息,并可能作为一个潜在的治疗靶标。
第二章NDRG1编码蛋白在胆囊癌细胞系GBC-SD中的表达
目的探讨NDRG1编码蛋白在胆囊癌细胞系GBC-SD中的表达
方法免疫细胞化学染色：应用特异性识别NDRG1编码蛋白的NDRG1多克隆抗体检测其在胆囊癌细胞系GBC-SD的表达。免疫印迹：应用NDRG1多克隆抗体验证NDRG1编码蛋白在该细胞系中的表达。
结果免疫细胞化学染色显示NDRG1蛋白在胆囊癌细胞系GBC-SD中呈阳性表达,兔疫印迹检测证实了这一发现,但NDRG1在该细胞系中表达较弱。
结论NDRG1蛋白在胆囊癌细胞系GBC-SD中呈阳性表达。
第三章NDRG1基因在胆囊癌细胞中的作用机制
目的采用基因转染过表达策略,通过对下游基因和表型的改变(主要包括细胞增殖、侵袭和凋亡三个方而)进行观察和分析NDRG1基因在胆囊癌细胞中表达情况及其与肿瘤生长、细胞增殖和细胞周期变化的关系。
方法质粒扩增及鉴定：大肠杆菌DH5α转化法扩增包含NDRG1完整开放阅读框架(ORF)的质粒pEGFP-NDRG1-N3和空载质粒pEGFP-N3,核酸内切酶Bam HI和KPnl双酶切和基因测序进行鉴定。质粒转染：以脂质体LipofectamineTM2000将上述质粒瞬时转染至胆囊癌细胞系GBC-SD。MTT检测：检测转染和未转染细胞增殖状况。流式细胞术：检测转染和未转染细胞细胞周期和凋亡。Transwell实验：检测转染和未转染细胞的迁移和侵袭。过河实验：检测转染和未转染细胞的迁移。免疫印迹：应用多种特异性抗体检测相关蛋白在转染和未转染细胞中的表达。
结果扩增后双酶切及测序鉴定表明扩增结果正确。转染pEGFP-NDRG1-N3后细胞呈现明显增殖加速和细胞周期演进、迁移和侵袭增强及表阿霉素所诱导细胞凋亡的显著减轻。这些表型出现的同时伴有c-fos、c-jun、cyclinD1、MMp-2、MMp-9、uPA和Bcl-2表达上调及p16、P27、Bid和剪切型caspase-3表达下调。而转染pEGFP-N3质粒后及未转染细胞则无类似效应。
结论NDRG1基因可促进胆囊癌细胞增殖、迁移和侵袭并可抑制其凋亡。这些作用可通过调节其下游多种基因的表达而实现。因此,NDRG1基因在胆囊癌中具有重要意义并有可能作为新的治疗靶点。
BACKGROUND
Primary gallbladder carcinoma ranked first in malignant tumors of biliary system, and ranked fifth in malignant tumors of digestive tract. The degree of malignancy of gallbladder carcinoma is very high. Because of the early nonspecific clinical manifestations, many patients at the time of diagnosis has progressed to the late, and5-year survival rate of stageⅢand Ⅳpatients is only1%and5%. The5-year survival rate of gallbladder cancer in United States and Europe is only5%-13%.The main treatment for gallbladder carcinoma is surgery, but only20%-30%of patients can be radical resection.The fact that chemotherapy and radiotherapy for gallbladder carcinoma are not sensitive has brought great difficulties to the clinical treatment for gallbladder carcinoma. It is important to explore new therapy.
N-myc downstream regulated gene1(NDRG1) was coloned from colon carcinoma cell line HT29-D4in the course of inducing differentiation by Van Belzen in1997. The gene can express in many tissues, such as kidney,heart,brain,bone muscle, placenta, lung, prostate, small intestine and ovary. But in leucocyte,immunity organ and liver, etc,its expression was low. The universality expression of NDRG1indicated that this gene might have important role in the course of histiocytic growth and differentiation.
Kurdistani found that NDRG1was low expressed in many tumor cell lines and carcinoma tissues, such as breast carcinoma, prostae carcinoma and colon carcinoma. Some scholars also proved that high expression of NDRG1could inhibit tumor gorwth. But some researchers indicated that the expression of NDRG1was high in many tumors. Cangul founded that in many cancers, such as lung caner, brain cancer, melanin tumor, liver cancer, prostate cancer and kidney cancer, the protein expression of NDRG1was high than normal control group. And this is due to the severe hypoxia in cancer tissues. So, NDRG1can be a marker of tissue hypoxia.
The exact role NDRG1plays in cancer metastasis is, however, still largely unclear. Specifically, there has been no report on the relation and mechanism between NDRG1gene expression and the biological behavior of primary gallbladder carcinoma in either Chinese literature or elsewhere.
Using two experimental models, resected clinical spceimens of human primary gallbladder carcinoma and established gallbladder carcinoma cell lines, this study investigates the impact of NDRG1expression on the metastatic potential of primary gallbladder carcinoma in an effort to identify NDRG1as a biomarker predicting early metastasis and a potential target for therapy.
The plan of this experiment is:In order to explore the expression of NDRG1in gallbladder carcinoma, and to find the role of NDRG1in gallbladder carcinoma occurring and development, to explore the effects of proteins about cell proliefartion on the expression of NDRG1and cell proliefartion in gallbladder carcinoma.This article detected integarlly the expression of NDRG1on tumor tissues and tumor-adjacent nomral mucosa in the141cases of gallbladder carcinoma by the immunohistochemistry and Western Blot. We detected the expression of NDRG1protein in the gallbladder carcinoma GBC-SD cells by immunohistochemistry and Western Blot methods. Designing and constructing pEGFP-NDRG1-N3and transfected it to GBC-SD cells by liposome and detecting the expression of NDRG1, changes of cell proliferation and cell cycle distribution.
Through above tests, we try to describe the character of NDRG1expression in gallbladder carcinoma and the function in tumor redifferentiation, and the relationship in NDRG1expression and cell proliferation and cell cycle disrtibution, and to provide new theory for the treatment of gallbladder carcinoma.
Part I Expression of N-myc downstream regulated gene1in primary gallbladder carcinoma and its correlation with clinicopathological features and clinical outcome
Objective N-myc downstream regulated gene1(NDRG1), a member of the N-myc downstream regulated gene family, is induced under a wide variety of stress and cell growth regulatory conditions. In several cancers, recent studies have shown its association with inhibition of tumor metastasis and suggested it to be a tumor suppressor gene. However, its significance in gallbladder carcinoma has not been studied. Therefore, the aim of this study was to investigate NDRG1expression in gallbladder carcinoma and its prognostic significance.
Methods We examined NDRG1expression in tumor specimens from141patients with gallbladder carcinoma by immunohistochemistry and analyzed the correlation between NDRG1expression and clinicopathologic factors or survival.
Results NDRG1was expressed in63.8%of gallbladder carcinoma but not in the normal epithelium of the gallbladder, remarkably at the invasive front of the tumors. In addition, NDRG1expression was significantly associated with high histologic grade, advanced pathologic T stage and clinical stage, positive nodal metastasis and venous/lymphatic invasion. Moreover, Kaplan-Meier curves showed that NDRG1 over-expression was significantly related to poor overall and disease-free survival (both p=0.02). Furthermore, multivariate analyses showed that NDRG1expression (hazard ratio,3.338; P=0.02) and clinical stage (hazard ratio,3.128; P=0.03) were independent risk factors for disease-free survival.
Conclusion Our data demonstrate for the first time that NDRG1expression in gallbladder carcinoma was significantly correlated with unfavorable clinicopathologic features and an independent poor prognostic factor for disease-free survival in patients. Taken together, our findings suggest that NDRG1expression could be used as a novel prognostic factor for patient survival and might be a potential therapeutic target in gallbladder carcinoma.
Part Ⅱ Expression of NDRG1eneoded proteins in gallbladder carcinoma cell line GBC-SD
Objective To investigate NDRG1expression in gallbladder carcinoma cell line GBC-SD.
Methods Immunocytochemical staining:The expression of NDRG1protein is detected by polyclonal antibody NDRG1, that specifically recognizes the protein, in gallbladder carcinoma cell line GBC-SD. Western Blot:The expression of NDRG1eneoded proteins in gallbladder carcinoma cell line GBC-SD is confirmed using polyclonal antibody NDRG1.
Results Immunocytochemical staining reveals that NDRG1protein is positively expressed in gallbladder carcinoma cell line GBC-SD. Western Blot detection confirms this finding but simultaneously shows that its expression is a bit weak.
Conclusion NDRG1protein is positively expressed in gallbladder carcinoma cell line GBC-SD.
Part Ⅲ Mechanisms of NDRG1gene in gallbladder carcinoma cells
Objective Designing and constructing pEGFP-NDRGl-N3and transfected it to GBC-SD cells by liposome and detecting the expression of NDRG1, changes of cell proliferation and cell cycle distribution.
Methods Plasmid amplification and identification:The Plasmids pEGFP-NDRG1-N3containing the complete open reading frame(ORF) of NDRG1and pEGFP-N3are transformed into E.Coli DH5a and further amplified. The products are identified by digestion of endonucleases, BamHI and KpnI,and DNA sequencing.
Plasmid transfection:Aforementioned Plasmids are trainsiently transfected into gallbladder carcinoma cell line CBC-SD by liposome LipofectamineTM2000.
MTT assay:To detect proliferation of transfected and negative control cells.
Flow cytometry:To detect cell cycle distribution and apoptosis of transfected and negative control cells.
Transwell test:To detect migration and invasion of transfected and negative control cells.
Crossing river test:To detect migration of transfected and negative control cells.
Western Blot:To detect expression of related proteins in transfected and negative control cells, using many kinds of specific antibody.
Results Digestion of two endonucleases and DNA sequencing indicate correct amplification. Cells transfected with pEGFP-NDRGl-N3shows significantly accelerated proliferation and cell cycle progression, enhanced migration and invasion, and attenuated apoptosis induced by epirubicin. These phenotypes are accompanied by upregulated expression of c-fos,c-jun, cyclinDl、MMp-2、MMp-9、uPA and Bcl-2, and downregulated expression of P16, P27, Bid and cleaved caspase-3. However, no similar effects are presented in cells transfected with the pEGFP-N3plasmid and negative control cells.
Conclusion NDRG1gene promotes proliferation, migration and invasion, and inhibits apoptosis of gallbladder carcinoma cells. These effects are achieved via regulation of expression of multiple downstream genes. Therefore, NDRG1gene is of important implications in gallbladder carcinoma and may be a novel therapeutic target.
引文
[I]Yang JM,Vassil AD, Hait WN. Activation of phospholipase C induces the expression of the multidrug resistance(MDR1) gene through the Raf-MAPK pathway[J]. Mol. Pharmacol.1996,60:674-80.
[2]Qayyum A, Mujtaba I. Effects of chemotherapy on patients with unresectable or metastatic adenocarcinoma of gallbladder[J]. JpakMed Assoc,2007,57(2):71-74.
[3]Young LC, Camp ling BG, Cole SP, et al.Mulitidrug resistance proteins MRP3, MRP1, MRP2 in lung cancer[J]. Chin Cancer Res,2001,7(6):1798-1840.
[4]Shi JS,Wang J. Progress in chemotherapy of cancer of bile ducts [J]. PostMed,2001,24(4):8-10.
[5]Misra S, Chaturvedi A, Misra NC, et al. Carcinoma of the gallbladder[J]. Lancet Oncol 2003,4:167-176.
[6]Melotte V, Qu X, Ongenaert M, et al. The N-myc downstream regulated gene (NDRG) family:diverse functions, multiple applications[J]. FASEB J.2010,24: 4153-66.
[7]Matsugaki T, Zenmyo M, Hiraoka K,et al. N-myc downstream-regulated gene 1/Cap43 expression promotes cell differentiation of human osteosarcoma cells[J]. Oncol Rep.2010,24:721-5.
[8]Jiang K, Shen Z, Ye Y, et al. A novel molecular marker for early detection and evaluating prognosis of gastric cancer:N-myc downstream regulated gene-1 (NDRG1) [J]. Scand J Gastroenterol.2010,45:898-908.
[9]Kitowska A, Pawelczyk T. N-myc downstream regulated 1 gene and its place in the cellular machinery [J]. Acta Biochim Pol.2010,57:15-21.
[10]Fan C, Yu J, Liu Y, et al. Increased NDRG1 Expression is Associated with Advanced T Stages and Poor Vascularization in Non-small Cell Lung Cancer[J]. Pathol Oncol Res.2010 In press.
[11]Jung EU, Yoon JH, Lee YJ, et al. Hypoxia and retinoic acid-inducible NDRG1 expression is responsible for doxorubicin and retinoic acid resistance in hepatocellular carcinoma cells[J]. Cancer Lett.2010,298:9-15.
[12]Nagai MA, Gerhard R, Fregnani JH, et al. Prognostic value of NDRG1 and SPARC protein expression in breast cancer patients[J]. Breast Cancer Res Treat. 2011,126:1-14.
[13]Tschan MP, Shan D, Laedrach J, et al. NDRG1/2 expression is inhibited in primary acute myeloid leukemia[J]. Leuk Res.2010,34:393-8.
[14]Akiba J, Ogasawara S, Kawahara A, et al. N-myc downstream regulated gene 1 (NDRG1)/Cap43 enhances portal vein invasion and intrahepatic metastasis in human hepatocellular carcinoma[J]. Oncol Rep.2008,20:1329-35.
[15]Li Q, Chen H. Transcriptional silencing of N-Myc downstream-regulated gene 1 (NDRG1) in metastatic colon cancer cell line SW620[J]. Clin Exp Metastasis. 2011,28:127-35.
[16]Ando T, Ishiguro H, Kimura M, et al. Decreased expression of NDRG1 is correlated with tumor progression and poor prognosis in patients with esophageal squamous cell carcinoma[J]. Dis Esophagus.2006,19:454-8.
[17]Liu W, Iiizumi-Gairani M, Okuda H, et al. KAI1 Gene Is Engaged in NDRG1 Gene-mediated Metastasis Suppression through the ATF3-NF{kappa}B Complex in Human Prostate Cancer[J]. J Biol Chem.2011,286:18949-59.
[18]Angst E, Dawson DW, Stroka D,et al. N-myc downstream regulated gene-1 expression correlates with reduced pancreatic cancer growth and increased apoptosis in vitro and in vivo[J]. Surgery.2011,149:614-24.
[19]Murakami Y, Hosoi F, Izumi H,et al. Identification of sites subjected to serine/threonine phosphorylation by SGK1 affecting N-myc downstream-regulated gene 1 (NDRG1)/Cap43-dependent suppression of angiogenic CXC chemokine expression in human pancreatic cancer cells[J]. Biochem Biophys Res Commun.2010,396:376-81.
[20]Chua MS, Sun H, Cheung ST,et al. Overexpression of NDRG1 is an indicator of poor prognosis in hepatocellular carcinoma[J]. Mod Pathol.2007,20:76-83.
[21]Zhao G, Chen J, Deng Y,et al. Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells[J]. Biochem Biophys Res Commun.2011,408:154-9.
[22]Aftab DT,Yang JM, Hait WN. Functional role of phosphorylation of the multidrug transporter(P-glycoprotein) by protein kinase C in multidrug-resiststant MCF-7cells[J].Oncol Res.1994,659-701.
[23]van Belzen N, Dinjens WN, Diesveld MP, et al. A novel gene which is up-regulated during colon epithelial cell differentiation and down-regulated in colorectal neoplasms[J]. Lab Invest,1997,77(1):85-92.
[24]Guan RJ, Ford HL, Fu Y, et al. Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer[J]. Cancer Res,2000,60 (3):749-755.
[25]Kokame K, Kato H,Miyata T. Nonradioactive differential display cloning of genes induced by homocysteine in vascular endothelial cells[J]. Methods,1998, 16(4):434-443.
[26]Kurdistani SK,Arizti P, Reimer CL, et al. Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to P53 and DNA damage[J]. Cancer Res,1998, 58(19):4439-4444.
[27]Piquemal D, Joulia D, Balaguer P, et al. Differential expression of the RTP/Drgl/Ndrl gene product in proliferating and growth arrested cells[J]. Biochim BioPhysActa,1999,1450(3):364-373.
[28]Bandyopadhyay S, Pai SK, Gross SC, et al. The Drg-1 gene suppresses tumor metastasis in prostate cancer[J].Cancer Res,2003,63(8):1731-1736.
[29]Xu B, Lin L, Rote NS. Identification of a stress-induced protein during human trophoblast differentiation by differential display analysis[J]. Biol Reprod, 1999,61(3):681-686.
[30]Agarwala KL, Kokame K, Kato H, et al. Phosphorylation of RTP, an ER stress-responsive cytoplasmic protein[J]. Biochem biophys Res Commun,2000, 272(3):641-647.
[31]Lin TM, Chang C. Cloning and charaeterization of TDD5, an androgen target gene that is differentially repressed by testosterone and dihydrotestosterone[J]. Proc Natl Acad Sci USA,1997,94(10):4988-4993.
[32]Park H, Adams MA, Lachat P, et al. Hypoxia induces the expression of a 43-kDa protein(PROXY-1) in normal and malignant cells[J]. Biochem Biophys Res Commun,2000,276(1):321-328.
[33]Nevin JE, Moran TJ, Kay S, et al. Carcinoma of the gallbladder:Staging, treatlnent, and Prognosis[J]. Cancer,1976,37:141-148.
[34]Kokame K, Kato H, Miyata T. Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis. GRP78/BiP and novel genes[J].J Biol Chem.1996,271(47):29659-65
[35]Wakisaka Y,Furuta A,Masuda K,et al.Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development[J]. J Histochem Cytochem.2003 Nov,51(11):1515-25.
[36]Kyuno J, Fukui A, Michiue T, et al. Identification and characterization of Xenopus NDRG1[J]. Biochem Biophys Res Commun.2003 Sep 12,309(1):52-7.
[37]Shimono A, Okuda T, Kondoh H. N-myc-dependent repression of ndrl, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant[J]. Mech Dev.1999 May,83(1-2):39-52.
[38]Song Y, Oda Y, Hori M,et al. N-myc downstream regulated gene-1/Cap43 may play an important role in malignant progression of prostate cancer, in its close association with E-cadherin[J]. Hum Pathol.2010,41:214-22.
[39]Liu YL, Bai WT, Luo W,et al. Downregulation of NDRG1 promotes invasion of human gastric cancer AGS cells through MMP-2[J]. Tumour Biol.2011,32: 99-105.
[40]Angst E, Sibold S, Tiffon C, et al. Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer [J]. Br J Cancer.2006,95:307-13.
[41]Wang Z, Wang F, Wang W-Q, et al. Correlation of N-myc downstream related gene 1 overexpression with progressive growth of colorectal neoplasm[J]. World J Gastroenterol.2004,10:550-4.
[42]Koyama S, Yoshioka T, Mizushima A, et al. Establishment of a cell line(G-415) from a human gallbladder carcinoma[J]. JPn J Cancer Res,1980,71:574-575.
[43]Knuth A, Gabbert H, Dippold W, et al. Biliary adenocarcinoma: charaeterisation of three new human tumor cell lines[J]. J Hepatol,1985, 1:579-596.
[44]Nakano S, Tatsumoto T, Esaki T, et al. Characterization of a newly established human gallbladder carcinoma cell line[J]. In Vitro Cell Dev Biol Anim,1994, 30A:729-732.
[45]Nishida T, Iwasasi H, Johzaki H, et al. A human gallbladder signet ring cell carcinoma cell line. Pathol Int,1997,47:368-376.
[46]刘博,王占民,吴小鹏,等.人胆囊癌细胞系胆囊癌-SD的建立[J]。山东医科大学学报,2000,38：332-333.
[47]刘博,王占民,吴小鹏,等.胆囊癌细胞系胆囊癌-SD的生物学行为研究[J]。外科理论与实践,2001,6：146-148.
[48]李东华,王占民,刘军,等.人胆囊癌细胞系胆囊癌-SD的多药耐药机制[J]。中华实验外科杂志,2003,20：995-997.
[49]乌新林,工占民,马道新,等.人胆囊癌细胞系胆囊癌-SD p53基因突变的研究[J]。山东大学学报·医学版,2005,43：66-68.
[50]乌新林,施琳,罗力,等.稳定表达外源野型生p53基因人胆囊癌细胞系的 建立和鉴定[J]。内蒙古医学院学报,2005,27：189-192.
[51]乌新林,王占民,杨凤辉,等.野生型p53基因对人胆囊癌细胞生长及致瘤性的抑制作用[J]。中国肿瘤生物治疗杂志,2005,12：266-271.
[52]刘颖斌,何小伟,王建伟,等.胆囊癌肝转移模型的建立和高转移细胞亚群的筛选[J]。中华医学杂志,2006,86：2117-2121.
[53]Chang XZ, Wang ZM, Yu JM, et al. Isolation of a human gallbladder cancer cell clone with high invasive phenotype in vitro and metastatic potential in orthotopic model and inhibition of its invasiveness by heparanase antisense oligodeoxynucleotides[J]. Clin Exp Metastasis,2007,24:25-38.
[54]冯立民,王建立,乌新林,等.单独及联合转染survivin和bcl-2反义寡核甘酸诱导胆囊癌细胞凋亡的研究[J]。中国肿瘤生物治疗杂志,2004,11：248-251.
[55]Ai Z, Lu W, Ton S, et al.Arsenic trioxide-mediated growth inhibition in gallbladder carcinoma cells via down-regulation of Cyclin D1 transcription mediated by SP1 transcription factor[J]. Biochem Biophys Res Commun,2007, 360:684-689.
[56]Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells[J]. Cancer Res,1987,47(12): 3239-3245.
[57]Junger WG, Cardoza TA, Liu FC, et al. Improved rapid photometric assay for quantitative measurement of PMN migration[J]. J Immunol Methods,1993, 160(1):73-79.
[58]范跃祖,傅锦业,赵泽明,等.去甲斑鳌素对人胆囊癌胆囊癌-SD细胞系增殖及侵袭的影响[J]。中华肿瘤杂志,2004,26：271-274.
[59]何静,邵根泽,周柔丽.肝癌中高表达的新基因LAPTM4B对细胞增殖及成瘤性的影响[J].北京大学学报(医学版),2003,35：348-352.
[60]王震.NDRGI基因与乳腺癌生物学行为相关性及其机制研究[D].上海：复旦大学,2006：26-46.
[61]Peng C, Zhou RL, Shao GZ, et al. Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its status of hepatocellular carcinoma[J]. Worid J Gastroenterol,2005,11:2704-2708.
[62]Oster SK, Ho CS, Soucie EL, et al. The myc oneogene:Marvelously complex[J]. Ady Cancer Res,2002,84:81-154.
[63]Milde-Langosch K. The Fos family of transcription factors and their role in tumourigenesis[J]. Eur J Cancer,2005,41:2449-2461.
[64]van Dam H, Castellazzi M. Distinct roles of Jun:Fos and Jun:ATF dimers in oncogenesis[J]. Oncogene,2001,20:2453-2464.
[65]Jochum W, Passegue E, Wagner EF. AP-1 in mouse development and tumourigenesis[J]. Oncogene,2001,20:2401-2412.
[66]Shaulian E, Karin M. AP-1 in cell proliferation and survival[J]. Oncogene,2001, 20:2390-2400.
[67]Shaulian E, Karin M.AP-1 as a regulator of cell life and death[J]. Nat Cell Biol, 2002,4:E131-E136.
[68]Jiang W, Kahn SM, Zhou P, et al. Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression[J]. Oncogene,1993,8:3447-3457.
[69]Ohtsubo M, Roberts JM. Cyclin-dependent regulation of G1 in mammalian fibroblasts[J]. Science,1993,259:1908-1912.
[70]Sherr CJ. G1 phaseprogression:Cycling on cue[J]. Cell,1994,79:551-555.
[71]Malumbres M, Barbacid M. Mammalian cyclin-dependent kinases[J]. TrendsBioehem Sci,2005,30:630-641.
[72]Canepa ET, Scassa ME, Ceruti JM, et al. INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions[J]. IUBMB Life, 2007,59:419-426.
[73]Gartel AL, shchors K. Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes[J]. Exp Cell Res,2003,283:17-21.
[74]Collier IE, Brans GA, Goldberg GI,et al. on the structure and chromosome location of the 72-and92-kDa human type IV collagenase genes[J].Genomics, 1991,9(3):429-434.
[75]Kleiner DE, Jr., Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteases[J]. Curr Opin Cell Biol,1993,5(5):891-897.
[76]MacDougall JR, Matrisian LM. Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis[J]. Cancer Metastasis ReV,1995,14(4):351-362.
[77]Autio-Harmainen H, Karttunen T, Hurskainen T, et al. Expression of 72 kilodalton type IV collagenase (gelatinase A) in benign and malignant ovarian tumors[J].Lab Invest,1993,69(3):312-321.
[78]Davies B, Miles DW, Happerfield LC,et al. Activity of type IV collagenases in benign and malignant breast disease[J]. Br J Cancer,1993,67(5):1126-1131.
[79]Stearns ME, Wang M. Type IVcollagenase(M(r)72,000) expression in human prostate:benign and malignant tissue[J]. Cancer Res,1993,53(4):878-883.
[80]D'Errico A, Garbisa S, Liotta LA, et al. Augmentation of type IV collagenase, laminin receptor, and Ki67 proliferation antigen associated with human colon, gastric, and breast earcinoma progression[J]. Mod Pathol,1991,4(2):239-246.
[81]Liabakk NB, Talbotl, Smith RA,et al. Matrix metalloprotease 2(MMP-2)and matrix metalloprotease 9(MMP-9) type Ⅳcollagenases in colorectal cancer[J]. Cancer Res,1996,56(1):190-196.
[82]Gullu IH, Kurdoglu M, Akalin I. The relation of gelatinase(MMP-2and-9) expression with distant site metastasis and tumour aggressiveness in colorectal cancer[J]. Br J Cancer 2000,82(1):249.
[83]Yan C Boyd DD. Regulation of matrix metalloproteinase gene expression[J]. J Cell Physiol,2007,211:19-26.
[84]Qin H, Sun Y, Benveniste EN. The transcription factors SP1,Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells[J]. J Biol Chen,1999,274:29130-29137.
[85]Mazzieri R, Masiero L, Zanetta L,et al. Control of type IVcollagenase activity by components of the urokinase-plasmin system:a regulatory mechanism with cell-bound reactants[J]. EMBO J,1997,16:2319-2332.
[86]Liu GY, Frank N, Bartsch H,et al. Induction of apoptosis by thiuramdisulfides, the reactive metabolites of dithiocarbamates, through coordinative modulation of NF kappaB, c-fos/c-jun,and P53 proteins[J]. Mol Carcinog,1998, 22:235-246.
[87]Takeuchi K, Motoda Y, Ito F. Role of transcription factor activator protein 1(AP1) in epidermal growth factor-mediated protection against apoptosis induced by a DNA-damaging agent[J]. FEBSJ,2006,273:3743-3755.
[88]Hemann MT, Lowe SW. The P53-Bcl-2 connection [J]. Cell Death Differ,2006, 13:1256-1259.
[89]Pelengaris S, Khan M. The many faces of c-MYC[J]. Arch Biochem Biophys, 2003,416:129-136.
[90]Conzen SD, Gottlob K, Kandel ES,et al. Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc:transrepression correlates with acceleration of apoptosis [J]. Mol Cell Biol,2000,20:6008-6018.
[1]Van Belzen N,Dinjens WN,Diesveld MP,et al. A novel gene which is up regulated during colon epithelial cell differentiation and down regulated in coloerctal neoplasms[J].Lab Invest,1997,77(l):85-92.
[2]Shimono A,Okuda T,KondohH,et al.N-myc-dependent repression of Ndrl,a gene identified by direct subtaretion of whole mouse embyro cDNAs bewteen wild type and N-myc mutant[J].Mech Dev,1999,83(1-2):39-52.
[3]Zhou D,Salnikow K,Costa M.Cap43, a novel gene specfically induced by Ni2+compounds[J]. Cancer Res,1998,58(10):2182-2189.
[4]Kurdistani SK,Arizti P,Reimer CL,et al.Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage [J]. Cancer Res, 1998,58(19):4439-4444.
[5]Pak H,Adams MA,Lachat P,et al. HyPoxia induce the expression of a 432kDa portein(PROXY21) in normal and malignant cells[J]. Biochem BioPhys Res Commun,2000,276(1):321-328.
[6]Kokame K,Kato H,Miyata T,et al.Nonradioactive differential display cloning of genes induced by homocysteine in vascular endothelial cells[J]. Methods,1998,16(4):434-443.
[7]Lin TM,Chang C.Cloning and characterization of TDD5,an andorgen target gene that 15 differentially repressed by tesosterone and dihydrotestosterone[J]. Proc Natl Acad Sci USA,1997,94(10):4988-4993.
[8]Yamauchi Y, Hongo S, Ohashi T, et al. Molecular cloning and characterization of a novel developmentally regulated gene, Bdml, showing predominant expression in postnatal rat brain[J]. Brain Res Mol Barin Res,1999,68 (1-2):149-158.
[9]Agarwala KL, Kokame K, Kato H, et al. Phosporylation of RTP, an ER sterss-responsive cytoplasmic protein[J].Biochem BioPhys Res Commun,2000, 272(3):641-647.
[10]Sugiki T, Murakami M,Taketomi Y,et al. N-myc down regulated gene 1 is a phosphoyrlated protein in mast cells[J]. Biol Pham Bull,2004,27(5):624-627.
[11]Wakisaka Y,Furuta A,Masuda K,et al.Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development.J Histochem Cytochem[J].2003 Nov,51(11):1515-25.
[12]Lachat P, Shaw P, Gebhard S, et al. Expression of NDRG1, a differentiation-related gene, in human tissues[J]. Histochem CellBiol,2002, 118(5):399-408.
[13]Piquemal D, Joulia D, Balaguer P, et al. Differential expression of the RTP/Drgl/Ndrl gene Product in Proliferating and growth arrested cells[J]. Biochim Biophys Acta,1999,1450(3):364-373.
[14]Hunter M,Bernard R,Freitas E,et al.Mutation screening of the N-myc downstream-regulated gene 1(NDRG1)in patients with Charcot-Marie-Tooth Disease[J]. Hum Mutat.2003 Aug,22(2):129-35.
[15]Kim KT,Ongusaha PP,Hong YK,et al. Function of Drgl/Rit42 in P53 dePendent mitotic spindle checkpoint[J]. J Biol Chem,2004,279(37):38597-38602.
[16]Kelly C,Chin AJ,Leatherman JL,et al.Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish[J]. Development.2000Sep,127(18):3899-911.
[17]Le NT, Richardson DR. lorn chelators with high antiproliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene:a link between iron metabolism and proliferation [J]. Blood,2004,104(9): 2967-2975.
[18]Ponka P.Iron and cell proliferation:another piece of the puzzle[J]. Blood 2004,104(9):2620-2621.
[19]Unoki M,Nakamura Y.Growth-suppressive effects of BPOZ and EGR2,two74 genes involved in the PTEN signaling pathway [J]. Oncogene.2001 Jul 27,20(33):4457-65.
[20]Kokame K,Kato H,Miyata T.Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis. GRP78/BiP and novel genes[J].J Biol Chem.1996,271(47):29659-65.
[21]Sato N,Kokame K,Shimokado K,et al.Changes of gene expression by lysophosphatidylcholine in vascular endothelial cells:12 up-regulated distinct genes including 5 cell growth-related,3 thrombosis-related, and 4 others[J]. J Biochem(Tokyo).1998 Jun,123(6):1119-26.
[22]Defacque H,Sevilla C,Piquemal D,et al.Potentiation of VD-induced monocytic leukemia cell differentiation by retinoids involves both RAR and RXR signaling pathways[J].Leukemia.1997 Feb,11(2):221-7.
[23]Guan RJ, Fodr HL, Fu Y, et al. Drg-1 as a differentiation related, putative metastatic suppressor gene inhuman colon cancer [J]. Cancer Res,2000, 60(3):749-755.
[24]何静,周柔丽.NDRG1基因与肝组织分化及癌变的相关性[J].北京大学学报(医学版),2003,35(5)：471-475.
[25]Akiba J,Ogasawara S,Kawahara A, eta.l N-myc downstream regulated gene 1 (NDRG1)/Cap43 enhancesportalvein invasion and intrahepatic metastasis inhuman hepatocellular[J]. carcinoma. Oncol Rep.2008,20(6):1329-1335.
[26]Cangul H.Hypoxia upregulates the expression of the NDRG1 gene leading to itsoverexpression in various human cancers[J].BMC Genet.2004 sep 2,5:27.
[27]张志强,何萍,姜又红.Cap43蛋白在人肺癌组织中的表达[J].癌变.畸变.突变.2007,19(6)：469-473.
[28]Wang Z,Wang F,Wang WQ,et al.Correlation of N-myc downstream regulated gene 1 overexpression with progressive growth of colorectal neoplasm[J].World JGastroenterol.2004 Feb 15,10(4):550-4.
[29]Song JY, Lee JK, Lee NW, et al. Microarray analysis of normal cervix carcinoma in situ, and invasive cervical cancenidentification of candidate genes in pathogenesis of invasion in cervical cancer[J].Int J Gynecol Cancer,2008, 18(5):1051-1059.
[30]Nishio S,Ushijima K,Tsuda N,et al.Cap43/NDRGl/Drg-l is a molecular target for angiogenesis and a prognostic indicator in cervical adenocarcinoma [J].Cancer Lett,2008,264(1):36-43.
[31]Nagai MA,Gerhard R,Fregnani JH,et al. Prognostic value of NDRG1 and SPARC protein expression in breast cancer patients[J].Breast Cancer Res Treat2010,12:3.
[32]Salnikow K, Kluz T, Costa M, et al. The regulation of hypoxic genes by calcium involves c-Jun/AP-1, which cooperates with hypoxia-inducible factor 1 in response to hypoxia. Mol Cell Biol,2002,22(6):1734-1741.
[33]Kurdistani SK,Rizti P,Reimer CL,et al.Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage[J].Cancer Res.1998 Oct 1,58(19):4439-44.
[34]Shah MA,Kemeny N,Hummer A,et al.Drgl expression in 131 colorectal liver metastases:correlation with clinical variables and patient outcomes[J].Clin Cancer Res.2005 May 1,11(9):3296-302.
[35]BandyoPadhyay S, Pai SK, Hirota S, et al. PTEN up-regulates the tumor metastasis suppressor gene Dgr-1 in Prostate and breast cancer [J]. Cancer Res, 2004,64(21):7655-7660.
[36]Wang Z,Liu Q,Chen Q,et al.Overexpression of NDRG1 relationship with proliferation activity and invasiveness of breast cancer cell line and breast cancer metastasis[J].Zhonghua Bing Li Xue Za Zhi.2006 Jun,35(6):333-8.
[37]Maruyama Y,Ono M,et al.Tumor growth suppression in pancreatic cancer by a putative metastasis suppressor gene Cap43/NDRG1/Drg-1 through modulation of angiogenesis[J].Cancer Res.2006 Jun 15,66(12):6233-42.
[38]Stein S,Thomas EK,Herzog B,et al. NDRG1 is necessary for P53-dependent apoptosis[J]. J Biol Chem,2004,279(47):48930-48940.
[39]Taketomi Y, Sugiki T, Saito T, et al. identification of NDRG1 as an early inducible gene during in vitro maturation of cultured mast cells[J]. Bioehem BioPhys Res Commun,2003,306(2):339-346.
[40]Salnikow K, Blagosklonny MV, Ryan H, et al. Cacrinogenic nickel induces genes involved with hypoxic stress[J]. Cnacer Res,2000,60(1):38-41.
[41]Cangul H, Salnikow K, Yee H, et al. Enhanced overexpression of an HIF-1/hypoxia-related protein in cancer cells[J]. Environ Health Perspect,2002, 110 Suppl 5:783-788.
[42]Salnikow K, Davidson T, Costa M. The role of hypoxia-inducible signaling pathway in nickel carcinogenesis[J]. Environ Health PersPect,2002,110 Suppl 5:831-834.
[43]Salnikow K,Davidson T,Zhang Q,et al. The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis[J]. Caneer Res,2003,63(13):3524-3530.
[44]Chen B, Nelson DM, Sadovsky Y. N-myc down-regulated gene 1 modulates the response of term human trophoblasts to hypoxic injury[J]. J Biol Chem,2006, 281(5):2764-2772.
[45]Segawa T, Nau ME, Xu LL, et al. Androgen-in duced expression of endoplasmic reticulum(ER) stress erspo nse genes in prostate cancer cells[J]. Oncogene,2002, 21(57):8749-8758.
[46]Malette B, Cherry E, Lagace M, et al. Large scale validation of human N-myc downstream-regulated gene (NDRG)-1 expression in endometrium during the menstrual cycle[J]. Mol Hum ReProd,2003,9(11):671-679.
[47]Ulrix W, Swinnen JV Heyns W, et al. The differentiation-related gene 1, Dgrl, is markedly upregulated by androgens in LNCaP prostatic adenocarcinoma cells[J]. FEBS Lett,1999,455(1-2):23-26.
[48]Kalyadjieva L, Gresham D, Gooding R, et al. N-myc downstream regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom[J].Am J Hum Genet,2000,67(1):47-58.
[49]Bennett CL,Chance PF.Molecular pathogenesis of hereditary motor,sensory and autonomic neuropathies[J].Curr Opin Neurol.2001 Oct,14(5):621-7.
[50]Okuda T, Higashi Y, Kokame K, et al. NDRG1 deficient mice exhibit a Progressive demyelinating disorder of peripheral nerves[J]. Mol Cell Biol,2004, 24(9):3949-3956.
[51]Hirata K, Masuda K, Morikawa W, et al.N-myc downstream-regulated gene 1 expression in injured sciatic nerves[J].Glia,2004,47(4):325-334.
[52]Salnikow K,An WQMelillo G,et al.Nickel-induced transformation shifts the balance between HIF-1 and p53 transcription factors. Carcinogenesis[J].1999 Sep,20(9):1819-23.
[53]Huang C,Ma WY,Dong Z.Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+cells. Mol Cell Biol[J].1996 Nov,16(11):6427-35.
[54]Huang C,Schmid PC,Ma WY,et al.Phosphatidylinositol-3 kinase is necessary for 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and activated protein 1 activation[J].J Biol Chem.1997 Feb 14,272(7):4187-94.
[55]Jiang BH,Jiang G,Zheng JZ,et al.Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1.Cell Growth Differ[J].2001 Jul,12(7): 363-9.
[56]Aoki M,Schetter C,Himly M,et al.The catalytic subunit of phosphoinositide 3-kinase:requirements for oncogenicity[J].J Biol Chem.2000 Mar 3,275 (9):6267-75.
[57]Cantley LC,Neel BGNew insights into tumor suppression:PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway [J].Proc Natl Acad Sci U S A.1999 Apr 13,96(8):4240-5.
[58]Li J,Davidson G,Huang Y,et al.Nickel compounds act through phosphatidylinositol-3-kinase/Akt-dependent,p70(S6k)-independent pathway to induce80 hypoxia inducible factor transactivation and Cap43 expression in mouse epidermal C141cells[J]. Cancer Res.2004 Jan 1,64(1):94-101.
[59]Zoroddu MA,Kowalik-Jankowska T,Kozlowski H,et al.Ni(Ⅱ)and Cu(Ⅱ) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR. J Inorg Biochem[J].2001 Mar,84(1-2):47-54.
[60]Dalton ML,Gadson PF Jr,Wrenn RW,et al.Homocysteine signal cascade: production of phospholipids,activation of protein kinase C,and the induction of c-fos and c-myb in smooth muscle cells[J].FASEB J.1997 Jul,11(8):703-11.
[61]Salnikow K, Kluz T, Costa M. Role of Ca2+in the regulation of nickel-inducible Cap43 gene expression[J]. Toxicol Appl Pharmacol,1999,160(2):127-132.
[62]Gomez-Caseor E, Navarro M, Rodriguez-Puebla ML. Regulation of the differentiation-related gene Dgr-1 during mouse skin cacrinogenesis[J]. Mol Carcinog,2001,32(2):100-109.
[63]贺付成,高冬玲,李惠翔,等.1,25-(OH)2VitD3对食管癌细胞分化相关基因NDRG1表达的影响[J].中国现代医学杂志,2005,15(24)：3746-3748.
[64]贺付成,陈奎生,张云汉,等.佛波酯对食管癌细胞分化相关基因NDRG1表达影响的研究[J].中华消化杂志,2006,15(3),195-196.
[65]Li S,Chen J,YangZ, eta.l N-myc downstream-regulated gene 1 as a down-regulated target gene of PTEN in the controlling of tumouri-genesis in endometrioid carcinoma[J]. Indian JMedRes.2008,127(5):453-459.
[66]Chen J, Li S, Yang Z, et al. Correlation between NDRG1 and PTEN expression in endometrial carcinoma[J]. Cancer Sci,2008,99(4):706-710.
[67]Yoshizumi T, Ohta T, Ninomiya I, et al. Thiazolidinedione, a Peroxisome proliferator-activated receptor-gamma ligand, inhibits growth and metastasis of HT-29 humnan colon cancer cells through differentiation-Promoting effects[J]. IntJ Oncol,2004,25(3):631-639.
[68]王立玮,周东蕊,房汝敬,等.乳腺癌中NDRG-1基因甲基化状态研究[J].现代医学,2008,36(2)：79-83.
[69]Li J, Kretzner L. The growth-inhibitory NDRG1 gene is a Myc negative target in human neuroblastomas and other cell types with overexPressed N-or c-myc[J].Mol Cell Biochem,2003,250(1-2):91-105
[70]Cangul H, Salnikow K, Yee H, et al. Enhanced expression of a novel Protein in human cancer cells:a potential aid to cancer diagnosis[J]. Cell Biol Toxicol, 2002,18(2):87-96
[71]Van Belzen N,Dinjens WN,Eussen BH,et al.Expression of differentiation-related genes in colorectal cancer:possible implications for prognosis[J]. Histol Histopathol.1998,13(4):1233-42.
[72]庄利萍,薛希筠,高倩,等NDRG1基因与肿瘤的研究进展[J].实用癌症杂志,2009,24(2)：211-212.
