核干因子在前列腺癌中的表达及其作用机制的初步研究
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摘要
前列腺癌是欧美国家男性人群最常见的恶性肿瘤。在我国，随着人口老龄化的发展，饮食结构等因素的变化，前列腺癌发病率及死亡率呈明显上升趋势。前列腺癌已经成为一个重要的公共健康问题，前列腺癌对放疗、化疗均不敏感，激素剥夺疗法容易变为难治性前列腺癌，特别是对于局部及全身转移的进展期患者，基因治疗是一种有希望的治疗方法。前列腺癌基因治疗，从引起前列腺癌的致病基因入手，意在从根本上治疗肿瘤，已经成为研究的热点，各国科学家也在不断的探索新的靶基因。
核干因子(nucleostemin，NS)是新近发现的一个p53结合蛋白，研究认为NS基因表达于干细胞和肿瘤细胞，在已分化的成体组织中不表达，NS基因在维持干细胞和多种癌细胞的增殖和自我更新中发挥重要作用。NS则可能是干细胞和癌细胞中重要的共同调节基因，已经证明在前列腺癌中存在肿瘤干细胞(tumorstem cells，TSCs)，TSC与前列腺癌的发生发展密切相关，因此对NS基因功能及其调节途径的研究对前列腺癌的诊断与治疗有重要意义。然而，NS基因在前列腺癌中是否也存在表达，其在前列腺癌发生、增殖过程中的作用及作用机制如何，尚不清楚。因此，本研究将检测NS基因在前列腺癌中的表达，并利用RNA干扰及表达谱基因芯片技术初步探讨其作用机制。
第一部分前列腺癌组织及细胞系中Nucleostemin基因表达的检测
目的揭示NS基因在前列腺癌组织及细胞系中的表达水平，探讨NS基因水平与前列腺癌患者临床指标的关系，并作为后续研究的基础。方法利用RT-PCR、蛋白印迹及免疫组化等方法对前列腺癌组织及PC-3、LNCap及DU145细胞中NS基因的表达进行检测，并探讨NS基因表达水平与前列腺癌患者临床指标的关系。结果前列腺癌标本及PC-3、LNCap及DU145细胞中都表达NS基因，NS基因在前列腺癌中的表达水平显著高于前列腺增生中的水平，NS基因表达水平与前列腺癌的分化程度呈负相关，细胞分化程度越差，NS基因表达水平越高。结论前列腺癌中高表达NS基因，NS基因在前列腺癌的不良分化和恶性增殖中可能起着重要作用。
第二部分Nucleostemin基因表达水平下调对PC-3细胞增殖能力的影响
目的探讨NS基因与前列腺癌恶性增殖的关系，为前列腺癌的基因治疗探索新的靶基因。方法通过shRNA质粒表达载体沉默PC-3细胞中NS基因的表达，研究NS基因水平下调后PC-3细胞形态、周期、增殖能力及凋亡的变化。结果NS基因在PC-3细胞中的表达水平下调后，细胞胞膜边缘突起增多，更趋向于分化；S期细胞的百分率降低，G1期的百分率升高；细胞的体外增殖速率明显降低，在裸鼠体内致瘤能力、肿瘤的生长速度及肿瘤的最终体积和重量也明显降低，细胞凋亡增多。结论NS基因可能是前列腺癌PC-3细胞周期中G1／S检查点的一个重要的调节因子，在PC-3细胞恶性增殖中发挥重要作用，NS可能是前列腺癌基因治疗的理想靶基因之一。
第三部分PC-3细胞Nucleostemin基因下调后全基因组表达谱的变化分析
目的筛选与NS相互作用的基因及可能的信号通路，探讨NS基因在前列腺癌中的作用机制。方法利用表达谱基因芯片技术分析PC-3细胞NS基因下调后全基因组表达谱的变化，筛选与NS相互作用的差异基因及信号通路，并利用实时荧光定量PCR对重要差异基因进行验证。结果筛选出了219个差异表达的基因，这些基因涉及到细胞周期、细胞增殖、信号转导、细胞凋亡及细胞分化等多个方面，重要差异基因被实时荧光定量PCR进一步证实。NS基因下调后主要引起INK4家族基因(P15，P16，P18)的上调和cyclin D1及HDAC1基因的下调，主要作用点位于CDK4／6-Cyclin D和pRb-E2F1复合体。结论在基因水平上证实了NS基因是细胞周期G1／S检查点的重要调控因子，在含突变型p53的前列腺癌中，NS可能主要是通过抑制p15、p16、p18等INK4家族的肿瘤抑制基因促进肿瘤的发生发展。发现了许多NS所涉及的肿瘤相关基因，但目前的工作仍不能完全揭示NS的信号通路，仍需进一步的实验去发现和证实。
Prostate cancer is the most common primary malignant tumor among malepopulation in the European and American countries. In China, the incidence andmortality rate have increased significantly due to population aging, changes in lifestyles and other causes. Prostate cancer has become a world-wide health problemwhich is not so sensitive to radiotherapy and chemotherapy, and many cases willbecome hormone-refractory prostate cancer when treated with hormone deprivation.Therefore, the gene therapy is a promising method, especially for the patients withlocal or systemic metastasis. The gene therapy for the treatment of prostate cancer,which targets the virulence genes of prostate cancer to cure this diseasefundamentally, has become the focus of research and many Scientists are nowexploring the new target genes.
Recently, a p53-binding protein, named nucleostemin (NS) exclusively expressedin the stem cells and cancer cells but not in differentiated adult tissues and cells, hasbeen shown to be essential for the proliferation and self-renewal of stem and cancercells. NS may be the common controlling gene of stem and cancer cells. Tumor stemcells (TSCs) which are thought to be related to the carcinogenesis of prostate cancerhave been identified in prostate cancer. Therefore, studies on the function andpathway of NS are of great importance to the diagnosis and treatment of prostatecancer. However, it is currently unknown whether NS is expressed in prostate cancerand how this newly identified molecule involved in prostate cancer pathogenesis. Thepresent study will detect the expression level of NS in prostate cancer andinvestigate its mechanism by RNA interference and DNA microarray technology.
PartⅠDetection of NS expression in prostate cancer tissuesand cell lines
Objective To reveal the NS expression level in prostate cancer tissues and celllines and investigate the correlation between NS gene level and clinical variables ofprostate cancer, taking this part as a base for later study. Methods RT-PCR,western blot and immunohistochemistry were used to detect the NS mRNA andprotein level in prostate cancer tissues and PC-3, LNCap and DU 145 cells, analyzingthe correlation between NS protein level and clinical variables of prostate cancer.Results NS gene was expressed in all the detected samples and cell lines, the NSmRNA and protein level in prostate cancer tissues were significantly higher thanthose in BPH tissues. NS protein level was negatively correlated with the degree ofcell differentiation in prostate cancer tissues, the worse the differentiation, the higherthe NS protein level. Conclusion NS gene is highly expressed in prostate cancerand may be of great importance to the adverse differentiation and malignantproliferation of prostate cancer.
PartⅡThe effect on the proliferation of PC-3 cells afterdown-regulating the NS gene level
Objective To investigate the relationship between the NS gene and themalignant proliferation of prostate cancer and explore the new target gene for prostatecancer gene therapy. Methods NS-specific short-hairpin RNA (shRNA) expressionplasmid was used to transfect PC-3 cells. The changes of cell morphology, cell cycle,proliferation ability and apoptosis were studied after down-regulating the NS genelevel. Results The cells became larger and showed more pseudopodia, having atendency to differentiate, the detection of cell cycle showed a decrease of S stage andan increase of G1 stare, cell proliferation ability in vitro and tumorigenesis ability in nude mice were discounted, the final tumor volume and weight were also decreasedafter knocking down NS gene. Conclusion NS may act as an important G1/Sregulator to regulate the malignant proliferation of prostate cancer PC-3 cells. NSmay serve as an ideal therapeutic gene target for prostate cancer.
PartⅢGene profiling after knocking-down the expression of NSgene in PC-3 cells
Objective To screen the genes and possible signal transduction pathways withwhich NS interact and explore the mechanism of NS in pro.state cancer. MethodsOligonucleotide DNA microarray was used to screen the genome changes afterknocking down expression of NS in PC-3 cells and real-time quantitative PCR wasused to further confirm the important differentially expressed genes. Results 219differentially expressed genes were found and theses genes were involved in cellcycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation.Some important differentially expressed genes were further Certified using real-timequantitative PCR. INK4 family genes (P15, P16, P18) were up-regulated and cyclinD1, HDAC1 were down-regulated, the main action points were CDK4/6-Cyclin Dand pRb-E2F1 complexes. Conclusion NS is an important G1/S checkpointregulator and its regulatory activity is certified at the gene level. Many tumor-relatedgenes are found involving in the NS-regulating pathways. However, the present studystill can not completely reveal the NS signal pathways and more studies are needed tofurther discover and confirm other mechanism.
核干因子(nucleostemin，NS)是新近发现的一个p53结合蛋白，研究认为NS基因表达于干细胞和肿瘤细胞，在已分化的成体组织中不表达，NS基因在维持干细胞和多种癌细胞的增殖和自我更新中发挥重要作用。NS则可能是干细胞和癌细胞中重要的共同调节基因，已经证明在前列腺癌中存在肿瘤干细胞(tumorstem cells，TSCs)，TSC与前列腺癌的发生发展密切相关，因此对NS基因功能及其调节途径的研究对前列腺癌的诊断与治疗有重要意义。然而，NS基因在前列腺癌中是否也存在表达，其在前列腺癌发生、增殖过程中的作用及作用机制如何，尚不清楚。因此，本研究将检测NS基因在前列腺癌中的表达，并利用RNA干扰及表达谱基因芯片技术初步探讨其作用机制。
第一部分前列腺癌组织及细胞系中Nucleostemin基因表达的检测
目的揭示NS基因在前列腺癌组织及细胞系中的表达水平，探讨NS基因水平与前列腺癌患者临床指标的关系，并作为后续研究的基础。方法利用RT-PCR、蛋白印迹及免疫组化等方法对前列腺癌组织及PC-3、LNCap及DU145细胞中NS基因的表达进行检测，并探讨NS基因表达水平与前列腺癌患者临床指标的关系。结果前列腺癌标本及PC-3、LNCap及DU145细胞中都表达NS基因，NS基因在前列腺癌中的表达水平显著高于前列腺增生中的水平，NS基因表达水平与前列腺癌的分化程度呈负相关，细胞分化程度越差，NS基因表达水平越高。结论前列腺癌中高表达NS基因，NS基因在前列腺癌的不良分化和恶性增殖中可能起着重要作用。
第二部分Nucleostemin基因表达水平下调对PC-3细胞增殖能力的影响
目的探讨NS基因与前列腺癌恶性增殖的关系，为前列腺癌的基因治疗探索新的靶基因。方法通过shRNA质粒表达载体沉默PC-3细胞中NS基因的表达，研究NS基因水平下调后PC-3细胞形态、周期、增殖能力及凋亡的变化。结果NS基因在PC-3细胞中的表达水平下调后，细胞胞膜边缘突起增多，更趋向于分化；S期细胞的百分率降低，G1期的百分率升高；细胞的体外增殖速率明显降低，在裸鼠体内致瘤能力、肿瘤的生长速度及肿瘤的最终体积和重量也明显降低，细胞凋亡增多。结论NS基因可能是前列腺癌PC-3细胞周期中G1／S检查点的一个重要的调节因子，在PC-3细胞恶性增殖中发挥重要作用，NS可能是前列腺癌基因治疗的理想靶基因之一。
第三部分PC-3细胞Nucleostemin基因下调后全基因组表达谱的变化分析
目的筛选与NS相互作用的基因及可能的信号通路，探讨NS基因在前列腺癌中的作用机制。方法利用表达谱基因芯片技术分析PC-3细胞NS基因下调后全基因组表达谱的变化，筛选与NS相互作用的差异基因及信号通路，并利用实时荧光定量PCR对重要差异基因进行验证。结果筛选出了219个差异表达的基因，这些基因涉及到细胞周期、细胞增殖、信号转导、细胞凋亡及细胞分化等多个方面，重要差异基因被实时荧光定量PCR进一步证实。NS基因下调后主要引起INK4家族基因(P15，P16，P18)的上调和cyclin D1及HDAC1基因的下调，主要作用点位于CDK4／6-Cyclin D和pRb-E2F1复合体。结论在基因水平上证实了NS基因是细胞周期G1／S检查点的重要调控因子，在含突变型p53的前列腺癌中，NS可能主要是通过抑制p15、p16、p18等INK4家族的肿瘤抑制基因促进肿瘤的发生发展。发现了许多NS所涉及的肿瘤相关基因，但目前的工作仍不能完全揭示NS的信号通路，仍需进一步的实验去发现和证实。
Prostate cancer is the most common primary malignant tumor among malepopulation in the European and American countries. In China, the incidence andmortality rate have increased significantly due to population aging, changes in lifestyles and other causes. Prostate cancer has become a world-wide health problemwhich is not so sensitive to radiotherapy and chemotherapy, and many cases willbecome hormone-refractory prostate cancer when treated with hormone deprivation.Therefore, the gene therapy is a promising method, especially for the patients withlocal or systemic metastasis. The gene therapy for the treatment of prostate cancer,which targets the virulence genes of prostate cancer to cure this diseasefundamentally, has become the focus of research and many Scientists are nowexploring the new target genes.
Recently, a p53-binding protein, named nucleostemin (NS) exclusively expressedin the stem cells and cancer cells but not in differentiated adult tissues and cells, hasbeen shown to be essential for the proliferation and self-renewal of stem and cancercells. NS may be the common controlling gene of stem and cancer cells. Tumor stemcells (TSCs) which are thought to be related to the carcinogenesis of prostate cancerhave been identified in prostate cancer. Therefore, studies on the function andpathway of NS are of great importance to the diagnosis and treatment of prostatecancer. However, it is currently unknown whether NS is expressed in prostate cancerand how this newly identified molecule involved in prostate cancer pathogenesis. Thepresent study will detect the expression level of NS in prostate cancer andinvestigate its mechanism by RNA interference and DNA microarray technology.
PartⅠDetection of NS expression in prostate cancer tissuesand cell lines
Objective To reveal the NS expression level in prostate cancer tissues and celllines and investigate the correlation between NS gene level and clinical variables ofprostate cancer, taking this part as a base for later study. Methods RT-PCR,western blot and immunohistochemistry were used to detect the NS mRNA andprotein level in prostate cancer tissues and PC-3, LNCap and DU 145 cells, analyzingthe correlation between NS protein level and clinical variables of prostate cancer.Results NS gene was expressed in all the detected samples and cell lines, the NSmRNA and protein level in prostate cancer tissues were significantly higher thanthose in BPH tissues. NS protein level was negatively correlated with the degree ofcell differentiation in prostate cancer tissues, the worse the differentiation, the higherthe NS protein level. Conclusion NS gene is highly expressed in prostate cancerand may be of great importance to the adverse differentiation and malignantproliferation of prostate cancer.
PartⅡThe effect on the proliferation of PC-3 cells afterdown-regulating the NS gene level
Objective To investigate the relationship between the NS gene and themalignant proliferation of prostate cancer and explore the new target gene for prostatecancer gene therapy. Methods NS-specific short-hairpin RNA (shRNA) expressionplasmid was used to transfect PC-3 cells. The changes of cell morphology, cell cycle,proliferation ability and apoptosis were studied after down-regulating the NS genelevel. Results The cells became larger and showed more pseudopodia, having atendency to differentiate, the detection of cell cycle showed a decrease of S stage andan increase of G1 stare, cell proliferation ability in vitro and tumorigenesis ability in nude mice were discounted, the final tumor volume and weight were also decreasedafter knocking down NS gene. Conclusion NS may act as an important G1/Sregulator to regulate the malignant proliferation of prostate cancer PC-3 cells. NSmay serve as an ideal therapeutic gene target for prostate cancer.
PartⅢGene profiling after knocking-down the expression of NSgene in PC-3 cells
Objective To screen the genes and possible signal transduction pathways withwhich NS interact and explore the mechanism of NS in pro.state cancer. MethodsOligonucleotide DNA microarray was used to screen the genome changes afterknocking down expression of NS in PC-3 cells and real-time quantitative PCR wasused to further confirm the important differentially expressed genes. Results 219differentially expressed genes were found and theses genes were involved in cellcycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation.Some important differentially expressed genes were further Certified using real-timequantitative PCR. INK4 family genes (P15, P16, P18) were up-regulated and cyclinD1, HDAC1 were down-regulated, the main action points were CDK4/6-Cyclin Dand pRb-E2F1 complexes. Conclusion NS is an important G1/S checkpointregulator and its regulatory activity is certified at the gene level. Many tumor-relatedgenes are found involving in the NS-regulating pathways. However, the present studystill can not completely reveal the NS signal pathways and more studies are needed tofurther discover and confirm other mechanism.
引文
[1] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006 [J]. CA Cancer J Clin,2006, 56(2):106-130.
[2] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2007[J]. CA Cancer J Clin,2007, 57(1):43-66.
[3] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55(2):74-108
[4] 叶定伟．前列腺癌的流行病学及中国的发病趋势[J]．中华外科杂志，2006，44(6)：362-364．
[5] Tsai RY, Mckay RD. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells [J]. Genes Dev, 2002, 16(23):2991-3003.
[6] Sijin Liu, Ziwei Cai, Yajun Liu, et al. Role of Nucleostemin in the growth regulation of gastric cancer, liver cancer and other cancers [J]. World J Gastroenterol, 2004, 10(9):1246-1249.
[7] Reya T, Morrison S J, Clarke MF, et al. Stem cells, cancer, and cancer stem cells[J].Nature, 2001, 414(6859):105-111.
[8] Collins AT, Habib FK, Maitland NJ, et al. Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin expression[J]. J Cell Sci, 2001, 114(pt21):3865-3872.
[9] Richardson GD, Robson CN, Lang SH, et al. CD133, a novel marker for human prostatic epithelial stem cells [J]. J Cell Sci, 2004, 117(pt16):3539-3545.
[10] Maitland NJ, Collins AT. A tumour stem cell hypothesis for the origins of prostate cancer [J]. BJU Int, 2005, 96(9): 1219-1223.
[11] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J].Nature, 1998,391 (6669):806-811.
[12] Hannon GJ. RNA interference [J]. Nature, 2002, 418(6894):244-251.
[13] McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs [J]. Nat Rev Genet, 2002, 3(10):737-747.
[14] Rocheleau CE, Downs WD, Lin R, et al. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos [J]. Cell, 1997, 90(4):707-716.
[15] Paul CP, Good PD, Winer I, et al. Effective expression of small interfering RNA in human cells [J]. Nat Biotechnol, 2002, 20(5):505-508.
[16] Sui G, Soohoo C, Affarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells [J]. Proc Natl Acad Sci U S A, 2002,99(8):5515-5520.
[17] Liu SJ, Cai ZW, Liu YJ, et al. The Effect of Knocking-down Nucleostemin Gene Expression on the in vitro Proliferation and in vivo Tumorigenesis of HeLa cells [J]. J Exp Clin Cancer Res, 2004, 23(3):529-538.
[18] 张志宏，刘思金，吴长利，等．RNA干扰抑制膀胱肿瘤细胞BIU-87核干因子基因表达对其增殖的影响[J]．中华泌尿外科杂志，2006，(S1)：36-38．
[19] White K, Rifkin S, Hurban P, et al. Microarray analysis of Drosophila development during metamorphosis [J]. Science, 1999, 286(5447):2179-2184.
[20] Wilson M, Derisi J, Kristensen H, et al. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray by hybridization [J].Proc Natl Acad Sci USA, 1999, 96(22):12833-12838.
[21] De Risi JL, Lyer VR, Brown PO. Exploring the metabolic and genetic control of gene expression on a genomic scale [J]. Science, 1997, 278(5338):680-686.
[22] Cheung V, Morley M, Aguilar F, et al. Making and reading microarrays [J]. Nat Genet, 1999, 21(1 Suppl):15-19.
[23] Bernardi R, Pandolfi PR The nucleolus: at the stem of immortality [J].Nat Med,2003, 9(1):24-25.
[24] Yang HX, Jin GL, Meng L, et al. Screening and identification of proteins interacting with nucleostemin [J]. World J Gastroenterol, 2005,11 (31):4812-4814.
[25] Carroll AG, Voeller HJ, Suqars L, et al. p53 oncogene mutations in three human prostate cancer cell lines [J]. Prostate, 1993, 23(2):123-134.
[26] van Bokhoven A, Verella-Garcia M, Korch C, et al. Molecular characterization of human prostate carcinoma cell lines [J]. Prostate, 2003, 57(3):205-225.
[27] Geschwind DH, Ou J, Easterday MC, et al. A genetic analysis of neural progenitor differentiation [J]. Neuron, 2001, 29(2):325-339.
[28] Ivanova NB, Dimos JJ, Schaniel C, et al. A stem cell molecular signature [J].Science, 2002, 298(5593):601-604.
[29] Rarnalho-Santos M, Yoon S, Matsuzaki Y, et al. Transcriptional profiling of embryonic and adult stem cells [J]. Science, 2002, 298(5593):597-600.
[30] Tsai RY, McKay RD. A multistep, GYP-driven mechanism controlling the dynamic cycling ofnucleostemin [J]. J Cell Biol, 2005, 168(2):179-184.
[31] Dundr M, and Misteli T. Nucleolomics: an inventory of the nucleolus [J]. Mol Cell, 2002, 9(1):5-7.
[32] Misteli T. The concept of self-organization in cellular architecture [J]. J Cell Biol,2001,155(2):181-185.
[33] Baddoo M, Hill K, Wilkinson R, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection [J]. J Cell Biochem, 2003, 89(6): 1235-1249.
[34] Bematchez R, Belkacemi L, Rassart E, et al. Differential expression of membrane and soluble adenylyl cyclase isoforms in cytotrophoblast cells and syncytiotrophoblasts of human placenta [J]. Placenta, 2003, 24(6):648-657.
[35] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J]. Cell, 2003,113(5): 643-655.
[36] 刘思金，蔡子微，胡国法，等．NS基因克隆及人成骨肉瘤细胞和人胚胎肾细胞NS基因的表达[J]．牡丹江医学院学报，2003，24(2)：5-7．
[37] 钱晖，许文荣，王文兵，等．骨髓间充质干细胞和部分肿瘤细胞Nucleostemin基 因的表达[J]．中国生物化学与分子生物学报，2005，21(1)：8-13．
[38] 刘思金，蔡子微，劳为德，等．NS特异性siRNA真核表达载体的构建[J]．牡丹江医学院学报，2003；24(4)：1-4．
[39] Drayton S, Peters G. Immortalisation and transformation revisited [J]. Curr Opin Genet Dev, 2002,12(1): 98-104
[40] Du YC, Stillman B. Yphlp, an ORC-interacting protein: Potential links between cell proliferation control, DNA replication, and ribosome biogenesis [J]. Cell, 2002, 109(7): 835-848.
[41] Michael D, Oren M. The p53 and Mdm2 families in cancer [J]. Curr Opin Genet Dev, 2002,12(1): 53-59.
[42] Kamijo T, Weber JD, Zambetti G, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2 [J]. Proc Natl Acad Sci, 1998, 95(14):8292-8297.
[43] Tao W, Levine AJ. P 19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2 [J]. Proc Natl Acad Sci, 1999, 96(12): 6937-6941.
[44] Yang H, Zhang J, Wu J, et al. Preparation and characterization of monoclonal antibodies against nucleostemin, a protein that controls cell proliferation in stem cells and cancer cells [J]. Hybridoma (Larchmt), 2005, 24(1):36-41.
[45] Y Fan, Z Liu, S Zhao, et al. Nucleostemin mRNA is expressed in both normal and malignant renal tissues [J]. Brit J Cancer, 2006, 94(11): 1658-1662.
[46] Nevins JR. Toward an understanding of the functional complexity of the E2F and retinoblastoma families [J]. Cell Growth Differ, 1998, 9(8):585-593.
[47] Dyson N. The regulation of E2F by pRB-family proteins [J]. Genes Dev, 1998,12(15):2245-2262.
[48] 敖朝晖，强伯勤．多聚酶链式反应(PCR)技术[A]．见：卢圣栋．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999：458-481．
[49] 肖渝平，薛庆善．体外培养的基本原理与技术[A]．见：薛庆善．体外培养的 原理与技术[M]．第二版．北京：科学出版社，2001：21-158．
[50] 李尹雄 王绯．核酸的分离与纯化[A]．见：卢圣栋．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999，101-121．
[51] Sean Gallagher，Scoot E．免疫印迹和免疫检测[A]．见：马学军，舒跃龙等译．精编分子生物学实验指南[M]．第四版．北京：科学出版社，2005，407-416．
[52] 皱中之．免疫细胞化学技术[A]．见：薛庆善．体外培养的原理与技术[M]．第二版．北京：科学出版社，2001：349-358．
[53] 张彤，徐勇，张晓光，等．前列腺癌中PIM-1的表达及其临床意义[J]．中华泌尿外科杂志，2006，27(9)：621-623．
[54] 杨靖轩 卢圣栋．基因克隆技术[A]．见：卢圣栋主编．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999，233-282．
[55] 林澄涛，姜燕芳，阴彬等．同尾酶技术再构建疟疾多价重组DNA疫苗中的应用[J]．中国生物化学与分子生物学报，1999，15(6)：974-977．
[56] 姚玲玲，王家宁，黄永章，等．利用同尾酶技术构建pET15b-PEP1-CAT重组质粒[J]．郧阳医学院学报，2006，25(1)：23-25．
[57] 刘强远 缪时英．基因表达物的检测[A]．见：卢圣栋．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999：397-403．
[58] 保天然．活细胞的观察检测方法[A]．见：薛庆善．体外培养的原理与技术[M]．第二版．北京：科学出版社，2001：334-339．
[59] Sun Y, Kim H, Parker M, et al. Lack of suppression of tumor cell phenotype by over expression of TIMP-3 in mouse JB6 tumor cells identification of a transfectant with increased tumorigenicity and invasiveness [J]. Anticancer Res 1996, 16(1): 1-7.
[60] 孙俊，张玉勋，李晋红．细胞周期与细胞同步化[A]．见：薛庆善．体外培养的原理与技术[M]．第二版．北京：科学出版社，2001：257-265．
[61] Takagi S, Mcfadden ML, Humphreys RE, et al. Detection of 5-bromo-2-deoxyuridine (BruUrd) incorporation with monoclonalanti-BruUrd antibody after deoxyribonuclease treatment[J]. Cytometry, 1993, 14:640-648.
[62] Brown PO, Botstein D. Exploring the new world of the genome with DNA microarray[J]. Nature Genetics (suppl), 1999, 21 (1):33-37.
[63] Xiang CC, Chen Y. cDNA microarray technology and its application[J].Biotechnology Advances, 2000, 18(1):35-46.
[64] Schena M, Heller RA, Theriault TP, et al. Microarray:biotechnology's discoveryplatform for functional genomics[J]. Trends Biotechnol, 1998;16(7):301-306.
[65] Liu SJ, Zhang ZH, Zhang DQ, et al. Gene profiling after knocking-down expression of nucleostemin in Hela cells using oligonucleotide DNA microarray[J]. J Exp Clin Cancer Res, 2006, 25(4):575-583.
[66] Roth JA, Swisher SG, Meyn RE, et al. p53 tumor suppressor gene therapy for cancer[J]. Oncology, 1999, 13: 148-154.
[67] Downing SR, Russell PJ, Jackson P. Alterations of p53 are common in early stage prostate cancer[J]. Can J Urol 2003; 10(4): 1924-1933.
[68] Nevins JR. Toward an understanding of the functional complexity of the E2F and retinoblastoma families [J]. Cell Growth Differ, 1998, 9(8): 585
[69] Dyson N. The regulation of E2F by pRB-family proteins [J]. Genes Dev, 1998,12(15): 2245
[70] 龚建平．细胞周期调控的两大机制[A]．见：曾益新．肿瘤学[M]．第二版．北京：人民卫生出版社．2003，166-180．
[71] 贺骏，阮秋蓉，杨秀萍，等．Pin1和Cyclin D1在人类5种常见肿瘤中的表达及其意义[J]．中国组织化学与细胞化学杂志，2006，15(6)：608-612．
[72] Drobnjak M, Osman I, Scher HI, et al. Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone [J]. Clin Cancer Res, 2000, 6(5): 1891-1895.
[73] Hunter T, Pines J. Cyclins and cancer. Ⅱ : cyclin D and CDK inhibitors come of age [J]. Cell 1994; 79:573-582.
[74] Aver DE. Histone deacetylases: transcriptional repression with SINers and NuRDs [J]. Trends cell boil, 1999, 9(5):193-198.
[75] Takimoto R, Kato J, Terui T, et al. Augmentation of antitumor effects of p53 gene therapy by combination with HDAC inhibitor[J]. Cancer Boil Ther, 2005, 4(4):421-428.
[76] Yan Zhao, Jing Tan, Li Zhuang, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activationof proapoptotic protein Bim [J]. Pans, 2005,102(44):16090-16095.
[77]Morris EJ, Michaud WA, Ji JY, et al. Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo[J]. PLoS Genet, 2006, 2(1 l):e196. [78] Hosaka S, Nakatsura T, Tsukamoto H, et al. Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo [J]. Cancer sci, 2006, 97(7):623-632.
[79]Lee JH, Rho SB, Chun T. Programmed cell death 6 (PDCD6) protein interacts with death-associated protein kinase 1 (DAPk1): additive effect on apoptosis via caspase-3 dependent pathway [J]. Biotechnol Lett, 2005, 27(14): 1011-1015. [80] Prescott JE, Osthus RC, Lee LA, et al. A novel c-Myc-responsive gene, JPO1, participates in neoplastic transformation [J]. J Biol Chem, 2001, 276(51):48276-48284.
[81] Osthus RC, Prescott JE, Karim B, et al. The Myc target gene JPO1/CDCA7 is frequently overexpressed in human tumors and has limited transforming activity in vivo[J].Cancer Res, 2005, 65(13):5620-5627.
[82] Goto Y, Hayashi R, Muramatsu T, et al. JPO1/CDCA7, a novel transcription factor E2F1-induced protein, possesses intrinsic transcriptional regulator activity [J]. Biochim Biophys Acta, 2006, 1759(1-2):60-68.
[83] Cangul H.Hypoxia upregulates the expression of the NDRG1 gene leading to its overexpression in various human cancers [J]. BMC Genet, 2004, 5:27-38. [84] Segawa T, Nau ME, Xu LL, et al. Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells[J]. Oncogene, 2002, 21(57):8749-8758.
[85] Tu LC, Yan X, Hood L, et al. Proteomics Analysis of the Interactome of N-myc Downstream Regulated Gene 1 and Its Interactions with the Androgen Response Program in Prostate Cancer Cells[J]. Mol Cell Proteomics, 2007, 6(4):575-588.
[86] Caruso, RP, Levinson B, Melamed J, et al. Altered N-myc downstream-regulated gene 1 protein expression in African-American compared with Caucasian prostate cancer patients [J]. Clin Cancer Res, 2004, 10(1 Pt l):222-227.
[2] Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2007[J]. CA Cancer J Clin,2007, 57(1):43-66.
[3] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002 [J]. CA Cancer J Clin, 2005, 55(2):74-108
[4] 叶定伟．前列腺癌的流行病学及中国的发病趋势[J]．中华外科杂志，2006，44(6)：362-364．
[5] Tsai RY, Mckay RD. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells [J]. Genes Dev, 2002, 16(23):2991-3003.
[6] Sijin Liu, Ziwei Cai, Yajun Liu, et al. Role of Nucleostemin in the growth regulation of gastric cancer, liver cancer and other cancers [J]. World J Gastroenterol, 2004, 10(9):1246-1249.
[7] Reya T, Morrison S J, Clarke MF, et al. Stem cells, cancer, and cancer stem cells[J].Nature, 2001, 414(6859):105-111.
[8] Collins AT, Habib FK, Maitland NJ, et al. Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)-integrin expression[J]. J Cell Sci, 2001, 114(pt21):3865-3872.
[9] Richardson GD, Robson CN, Lang SH, et al. CD133, a novel marker for human prostatic epithelial stem cells [J]. J Cell Sci, 2004, 117(pt16):3539-3545.
[10] Maitland NJ, Collins AT. A tumour stem cell hypothesis for the origins of prostate cancer [J]. BJU Int, 2005, 96(9): 1219-1223.
[11] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J].Nature, 1998,391 (6669):806-811.
[12] Hannon GJ. RNA interference [J]. Nature, 2002, 418(6894):244-251.
[13] McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs [J]. Nat Rev Genet, 2002, 3(10):737-747.
[14] Rocheleau CE, Downs WD, Lin R, et al. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos [J]. Cell, 1997, 90(4):707-716.
[15] Paul CP, Good PD, Winer I, et al. Effective expression of small interfering RNA in human cells [J]. Nat Biotechnol, 2002, 20(5):505-508.
[16] Sui G, Soohoo C, Affarel B, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells [J]. Proc Natl Acad Sci U S A, 2002,99(8):5515-5520.
[17] Liu SJ, Cai ZW, Liu YJ, et al. The Effect of Knocking-down Nucleostemin Gene Expression on the in vitro Proliferation and in vivo Tumorigenesis of HeLa cells [J]. J Exp Clin Cancer Res, 2004, 23(3):529-538.
[18] 张志宏，刘思金，吴长利，等．RNA干扰抑制膀胱肿瘤细胞BIU-87核干因子基因表达对其增殖的影响[J]．中华泌尿外科杂志，2006，(S1)：36-38．
[19] White K, Rifkin S, Hurban P, et al. Microarray analysis of Drosophila development during metamorphosis [J]. Science, 1999, 286(5447):2179-2184.
[20] Wilson M, Derisi J, Kristensen H, et al. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray by hybridization [J].Proc Natl Acad Sci USA, 1999, 96(22):12833-12838.
[21] De Risi JL, Lyer VR, Brown PO. Exploring the metabolic and genetic control of gene expression on a genomic scale [J]. Science, 1997, 278(5338):680-686.
[22] Cheung V, Morley M, Aguilar F, et al. Making and reading microarrays [J]. Nat Genet, 1999, 21(1 Suppl):15-19.
[23] Bernardi R, Pandolfi PR The nucleolus: at the stem of immortality [J].Nat Med,2003, 9(1):24-25.
[24] Yang HX, Jin GL, Meng L, et al. Screening and identification of proteins interacting with nucleostemin [J]. World J Gastroenterol, 2005,11 (31):4812-4814.
[25] Carroll AG, Voeller HJ, Suqars L, et al. p53 oncogene mutations in three human prostate cancer cell lines [J]. Prostate, 1993, 23(2):123-134.
[26] van Bokhoven A, Verella-Garcia M, Korch C, et al. Molecular characterization of human prostate carcinoma cell lines [J]. Prostate, 2003, 57(3):205-225.
[27] Geschwind DH, Ou J, Easterday MC, et al. A genetic analysis of neural progenitor differentiation [J]. Neuron, 2001, 29(2):325-339.
[28] Ivanova NB, Dimos JJ, Schaniel C, et al. A stem cell molecular signature [J].Science, 2002, 298(5593):601-604.
[29] Rarnalho-Santos M, Yoon S, Matsuzaki Y, et al. Transcriptional profiling of embryonic and adult stem cells [J]. Science, 2002, 298(5593):597-600.
[30] Tsai RY, McKay RD. A multistep, GYP-driven mechanism controlling the dynamic cycling ofnucleostemin [J]. J Cell Biol, 2005, 168(2):179-184.
[31] Dundr M, and Misteli T. Nucleolomics: an inventory of the nucleolus [J]. Mol Cell, 2002, 9(1):5-7.
[32] Misteli T. The concept of self-organization in cellular architecture [J]. J Cell Biol,2001,155(2):181-185.
[33] Baddoo M, Hill K, Wilkinson R, et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection [J]. J Cell Biochem, 2003, 89(6): 1235-1249.
[34] Bematchez R, Belkacemi L, Rassart E, et al. Differential expression of membrane and soluble adenylyl cyclase isoforms in cytotrophoblast cells and syncytiotrophoblasts of human placenta [J]. Placenta, 2003, 24(6):648-657.
[35] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells [J]. Cell, 2003,113(5): 643-655.
[36] 刘思金，蔡子微，胡国法，等．NS基因克隆及人成骨肉瘤细胞和人胚胎肾细胞NS基因的表达[J]．牡丹江医学院学报，2003，24(2)：5-7．
[37] 钱晖，许文荣，王文兵，等．骨髓间充质干细胞和部分肿瘤细胞Nucleostemin基 因的表达[J]．中国生物化学与分子生物学报，2005，21(1)：8-13．
[38] 刘思金，蔡子微，劳为德，等．NS特异性siRNA真核表达载体的构建[J]．牡丹江医学院学报，2003；24(4)：1-4．
[39] Drayton S, Peters G. Immortalisation and transformation revisited [J]. Curr Opin Genet Dev, 2002,12(1): 98-104
[40] Du YC, Stillman B. Yphlp, an ORC-interacting protein: Potential links between cell proliferation control, DNA replication, and ribosome biogenesis [J]. Cell, 2002, 109(7): 835-848.
[41] Michael D, Oren M. The p53 and Mdm2 families in cancer [J]. Curr Opin Genet Dev, 2002,12(1): 53-59.
[42] Kamijo T, Weber JD, Zambetti G, et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2 [J]. Proc Natl Acad Sci, 1998, 95(14):8292-8297.
[43] Tao W, Levine AJ. P 19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2 [J]. Proc Natl Acad Sci, 1999, 96(12): 6937-6941.
[44] Yang H, Zhang J, Wu J, et al. Preparation and characterization of monoclonal antibodies against nucleostemin, a protein that controls cell proliferation in stem cells and cancer cells [J]. Hybridoma (Larchmt), 2005, 24(1):36-41.
[45] Y Fan, Z Liu, S Zhao, et al. Nucleostemin mRNA is expressed in both normal and malignant renal tissues [J]. Brit J Cancer, 2006, 94(11): 1658-1662.
[46] Nevins JR. Toward an understanding of the functional complexity of the E2F and retinoblastoma families [J]. Cell Growth Differ, 1998, 9(8):585-593.
[47] Dyson N. The regulation of E2F by pRB-family proteins [J]. Genes Dev, 1998,12(15):2245-2262.
[48] 敖朝晖，强伯勤．多聚酶链式反应(PCR)技术[A]．见：卢圣栋．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999：458-481．
[49] 肖渝平，薛庆善．体外培养的基本原理与技术[A]．见：薛庆善．体外培养的 原理与技术[M]．第二版．北京：科学出版社，2001：21-158．
[50] 李尹雄 王绯．核酸的分离与纯化[A]．见：卢圣栋．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999，101-121．
[51] Sean Gallagher，Scoot E．免疫印迹和免疫检测[A]．见：马学军，舒跃龙等译．精编分子生物学实验指南[M]．第四版．北京：科学出版社，2005，407-416．
[52] 皱中之．免疫细胞化学技术[A]．见：薛庆善．体外培养的原理与技术[M]．第二版．北京：科学出版社，2001：349-358．
[53] 张彤，徐勇，张晓光，等．前列腺癌中PIM-1的表达及其临床意义[J]．中华泌尿外科杂志，2006，27(9)：621-623．
[54] 杨靖轩 卢圣栋．基因克隆技术[A]．见：卢圣栋主编．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999，233-282．
[55] 林澄涛，姜燕芳，阴彬等．同尾酶技术再构建疟疾多价重组DNA疫苗中的应用[J]．中国生物化学与分子生物学报，1999，15(6)：974-977．
[56] 姚玲玲，王家宁，黄永章，等．利用同尾酶技术构建pET15b-PEP1-CAT重组质粒[J]．郧阳医学院学报，2006，25(1)：23-25．
[57] 刘强远 缪时英．基因表达物的检测[A]．见：卢圣栋．现代分子生物学实验技术[M]．第二版．北京：中国协和医科大学出版社，1999：397-403．
[58] 保天然．活细胞的观察检测方法[A]．见：薛庆善．体外培养的原理与技术[M]．第二版．北京：科学出版社，2001：334-339．
[59] Sun Y, Kim H, Parker M, et al. Lack of suppression of tumor cell phenotype by over expression of TIMP-3 in mouse JB6 tumor cells identification of a transfectant with increased tumorigenicity and invasiveness [J]. Anticancer Res 1996, 16(1): 1-7.
[60] 孙俊，张玉勋，李晋红．细胞周期与细胞同步化[A]．见：薛庆善．体外培养的原理与技术[M]．第二版．北京：科学出版社，2001：257-265．
[61] Takagi S, Mcfadden ML, Humphreys RE, et al. Detection of 5-bromo-2-deoxyuridine (BruUrd) incorporation with monoclonalanti-BruUrd antibody after deoxyribonuclease treatment[J]. Cytometry, 1993, 14:640-648.
[62] Brown PO, Botstein D. Exploring the new world of the genome with DNA microarray[J]. Nature Genetics (suppl), 1999, 21 (1):33-37.
[63] Xiang CC, Chen Y. cDNA microarray technology and its application[J].Biotechnology Advances, 2000, 18(1):35-46.
[64] Schena M, Heller RA, Theriault TP, et al. Microarray:biotechnology's discoveryplatform for functional genomics[J]. Trends Biotechnol, 1998;16(7):301-306.
[65] Liu SJ, Zhang ZH, Zhang DQ, et al. Gene profiling after knocking-down expression of nucleostemin in Hela cells using oligonucleotide DNA microarray[J]. J Exp Clin Cancer Res, 2006, 25(4):575-583.
[66] Roth JA, Swisher SG, Meyn RE, et al. p53 tumor suppressor gene therapy for cancer[J]. Oncology, 1999, 13: 148-154.
[67] Downing SR, Russell PJ, Jackson P. Alterations of p53 are common in early stage prostate cancer[J]. Can J Urol 2003; 10(4): 1924-1933.
[68] Nevins JR. Toward an understanding of the functional complexity of the E2F and retinoblastoma families [J]. Cell Growth Differ, 1998, 9(8): 585
[69] Dyson N. The regulation of E2F by pRB-family proteins [J]. Genes Dev, 1998,12(15): 2245
[70] 龚建平．细胞周期调控的两大机制[A]．见：曾益新．肿瘤学[M]．第二版．北京：人民卫生出版社．2003，166-180．
[71] 贺骏，阮秋蓉，杨秀萍，等．Pin1和Cyclin D1在人类5种常见肿瘤中的表达及其意义[J]．中国组织化学与细胞化学杂志，2006，15(6)：608-612．
[72] Drobnjak M, Osman I, Scher HI, et al. Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone [J]. Clin Cancer Res, 2000, 6(5): 1891-1895.
[73] Hunter T, Pines J. Cyclins and cancer. Ⅱ : cyclin D and CDK inhibitors come of age [J]. Cell 1994; 79:573-582.
[74] Aver DE. Histone deacetylases: transcriptional repression with SINers and NuRDs [J]. Trends cell boil, 1999, 9(5):193-198.
[75] Takimoto R, Kato J, Terui T, et al. Augmentation of antitumor effects of p53 gene therapy by combination with HDAC inhibitor[J]. Cancer Boil Ther, 2005, 4(4):421-428.
[76] Yan Zhao, Jing Tan, Li Zhuang, et al. Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activationof proapoptotic protein Bim [J]. Pans, 2005,102(44):16090-16095.
[77]Morris EJ, Michaud WA, Ji JY, et al. Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo[J]. PLoS Genet, 2006, 2(1 l):e196. [78] Hosaka S, Nakatsura T, Tsukamoto H, et al. Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo [J]. Cancer sci, 2006, 97(7):623-632.
[79]Lee JH, Rho SB, Chun T. Programmed cell death 6 (PDCD6) protein interacts with death-associated protein kinase 1 (DAPk1): additive effect on apoptosis via caspase-3 dependent pathway [J]. Biotechnol Lett, 2005, 27(14): 1011-1015. [80] Prescott JE, Osthus RC, Lee LA, et al. A novel c-Myc-responsive gene, JPO1, participates in neoplastic transformation [J]. J Biol Chem, 2001, 276(51):48276-48284.
[81] Osthus RC, Prescott JE, Karim B, et al. The Myc target gene JPO1/CDCA7 is frequently overexpressed in human tumors and has limited transforming activity in vivo[J].Cancer Res, 2005, 65(13):5620-5627.
[82] Goto Y, Hayashi R, Muramatsu T, et al. JPO1/CDCA7, a novel transcription factor E2F1-induced protein, possesses intrinsic transcriptional regulator activity [J]. Biochim Biophys Acta, 2006, 1759(1-2):60-68.
[83] Cangul H.Hypoxia upregulates the expression of the NDRG1 gene leading to its overexpression in various human cancers [J]. BMC Genet, 2004, 5:27-38. [84] Segawa T, Nau ME, Xu LL, et al. Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells[J]. Oncogene, 2002, 21(57):8749-8758.
[85] Tu LC, Yan X, Hood L, et al. Proteomics Analysis of the Interactome of N-myc Downstream Regulated Gene 1 and Its Interactions with the Androgen Response Program in Prostate Cancer Cells[J]. Mol Cell Proteomics, 2007, 6(4):575-588.
[86] Caruso, RP, Levinson B, Melamed J, et al. Altered N-myc downstream-regulated gene 1 protein expression in African-American compared with Caucasian prostate cancer patients [J]. Clin Cancer Res, 2004, 10(1 Pt l):222-227.