摘要
脑胶质瘤是人类中枢神经系统最常见的难治性恶性肿瘤之一,其病因、发病机制、有效治疗方法仍在探索之中。近些年来,人们越来越多地把目光投向胶质瘤的基因治疗。寻找与脑胶质瘤发病相关的基因,深入了解胶质瘤的分子病理,在此基础上寻找胶质瘤治疗的新策略和靶点,成为胶质瘤研究领域的热点问题。
p21WAF1/CIP,(p21)基因是细胞周期蛋白依赖性激酶(cyclin dependent kinase, cdk)抑制剂,是目前已知的具有最广泛活性的细胞周期抑制基因,参与了细胞增殖、分化、衰老和凋亡等多种功能的调节。Survivin是近期被发现的凋亡抑制蛋白(inhibitor of apoptosis protein, IAPs)家族成员。主要参与了细胞有丝分裂和凋亡过程。Survivin在肿瘤发生发展、患者预后中的重要作用,使其成为肿瘤综合治疗,尤其是基因治疗的重要靶点。
RNA激活(RNA activation, RNAa),是指某些小分子非编码RNA(non-coding RNA, ncRNA)在转录水平激活基因表达的现象。利用RNAa技术特异性激活肿瘤抑癌基因从而治疗肿瘤是肿瘤基因治疗的新思路和新方法。通过RNAa技术特异性上调p21基因的表达,抑制人肾癌,膀胱癌,肺癌,肝癌等肿瘤组织的生长,促进肿瘤细胞凋亡已经得到实验证实,但尚无RNAa在颅内胶质瘤中激活特定的抑癌基因达到治疗肿瘤目的的研究和报道。
本课题主要研究人脑胶质瘤细胞中p21和survivin基因的表达与组织学分级之间的关系；以及探讨应用saRNA靶向激活p21对人脑胶质瘤细胞系基因表达,细胞增殖,细胞凋亡和细胞周期分布产生的影响及相关机制,藉此探索临床抗胶质瘤治疗可能的新靶点及新方法。研究内容分为两个部分。
第一部分目的：探讨p21及survivin蛋白在人脑胶质瘤细胞中的定位及表达与肿瘤组织学分级之间的关系；方法：应用免疫组化SP法检测Ⅰ-Ⅳ级人脑胶质瘤组织中p21以及survivin蛋白的表达,并分析其与胶质瘤组织学分级之间的关系。结果：p21蛋白阳性染色定位于人脑胶质瘤细胞核中,为棕黄色或棕褐色,不同级别胶质瘤中,p21的阳性表达率不同,胶质瘤级别越高,p21表达水平越低。survivin蛋白阳性染色主要定位于人脑胶质瘤细胞浆内,呈淡黄、棕黄色或棕褐色着色,偶尔可见细胞核着色,不同级别胶质瘤中,survivin的阳性表达率不同,胶质瘤级别越高,survivin表达水平越高。结论：p2l蛋白阳性表达率随着肿瘤病理级别的增高而降低,即恶性程度越高,p2l阳性表达率越低。survivin蛋白阳性表达率随着肿瘤病理级别的增高而增高,即恶性程度越高,survivin阳性表达率越高。
第二部分目的：探讨应用saRNA激活p21表达对人脑胶质瘤细胞系基因表达,细胞增殖,凋亡和细胞周期分布的影响。方法：设计抑癌基因p21启动子DNA序列互补的双链RNA分子(dsP21),转染胶质瘤细胞系SHG-44,应用实时荧光定量PCR (RT-PCR)及蛋白质印迹法(Western blotting)检测p21基因、Survivin基因mRNA及蛋白质的表达变化；MTT法检测胶质瘤细胞增殖速度的变化；流式细胞仪检测胶质瘤细胞凋亡率和细胞周期分布的变化。结果：1.dsP21转染胶质瘤细胞72h后,RT-PCR和Western blotting结果显示,p21表达显著上调,survivin表达明显下调,空白对照组、阴性对照组和实验组p21mRNA、蛋白质,Survivin mRNA、蛋白质相对表达量比较差异显著,有统计学意义。2.dsP21转染胶质瘤细胞第三天后,胶质瘤细胞增殖受到明显抑制。3.dsP21转染胶质瘤细胞72h后细胞凋亡率明显增加,细胞周期分布显示G0/G1期细胞明显增多,S期细胞减少。结论：RNAa上调p21基因表达明显抑制了人脑胶质瘤细胞增殖,促进了胶质瘤细胞凋亡,使胶质瘤细胞阻滞在G0/G1期,抑制了人脑胶质瘤细胞survivin基因表达,具有明显的体外抗肿瘤效果；RNAa可以用来特异性激活肿瘤抑制基因治疗人脑胶质瘤。
Glioma is the most refractory malignancy in human central nervous system. The cause of disease, pathogenesis and the effective intervention of this tumor are still ambiguous. In recent years, people increasingly pay their attention to gene therapy of glioma. It is become the prevalent issue to pursue pathogenesis related genes, penetrate the molecular pathology and on this basis looking for new strategies and targets to treat glioma.
P21WAF1/CIP1(p21) gene is an inhibitor of cyclin-dependent kinase(cdk) and the most widely active gene of cell cycle inhibitor currently known which involved in variety of regulatory functions, such as cell proliferation, differentiation, aging and apoptosis. Survivin was discovered as a IAPs family members recently. Survivin involves in cell mitosis and apoptosis process. Survivin plays an important role in the progression of tumors and the prognosis of patients making it is a significant target for combined modality therapy, especially gene therapy in human'glioma.
RNA activation (RNAa), refers to describe the phenomenon of gene activation at the transcriptional level mediated by some small non-coding RNA(non-coding RNA, ncRNA) molecules. This is a new ideas and ways of gene therapies to treating tumors by utilizing RNAa to activate the tumor suppressor gene. It has been confirmed experimentally that the increased expression of p21by specifically activating gene expression may inhibit the growth of human renal carcinoma, bladder cancer, lung cancer, liver cancer and other tumors, but no study has been reported about intracranial glioma.
The purpose of this study is to explore the relationship between the expression of p21and survivin in glioma cells and the histological grade of glioma; as well as to investigate the effectiveness and related mechanisms about cell proliferation, apoptosis and cell cycle distribution by utilizing saRNA to activate the expression of p21, thereby to discuss the possible new target and new methods for clinical anti-glioma treatment. The study is divided into two parts.
The first part Aim:To investigate the expression and localization of p21and survivin protein, and the relationship between the expression of p21and survivin and histological grades in human glioma; Methods:The expression of p21and survivin of I-IV grade human glioma was examined with immunohistochemical technique. The relationship of the expression of p21and survivin with the histological grades of the glioma was analyzed. Results:The positive staining of p21protein positioned in the nuclei of the glioma cells, it was brown or tan. There were significant differences between the positive expression rates of p21and different grade of glioma. With higher malignancy, the expression level decreased. The positive staining of survivin protein positioned in the cytoplasm, occasionally been seen in nuclei of the glioma cells, it was pale yellow, brown, yellow or tan coloring. There were significant differences between the positive expression rates of survivin and different grade of glioma. With higher malignancy, the expression level increased. Conclusions:The decreased expression of p21in glioma was related to the malignancy of the tumor. The increased expression of survivin in glioma was related to the malignancy of the tumor.
The second part Aim:to investigate the effectiveness about cell proliferation, apoptosis and cell cycle distribution by utilizing saRNA to activate the expression of p21. Methods:Small double-stranded RNA molecules(dsP21) which complementary with p21promoter DNA sequences was designed to transfect glioma cell line SHG-44. Real-time PCR and Western blot analysis were conducted to detect p21and survivin mRNA and protein respectively. Cell proliferation was examined by MTT assay. Apoptosis and cell cycle distribution were detected by flow-cytometric analysis. Results:1. The up-regulated expression of p21mRNA and protein were statistically significant and the down-regulated expression of survivin mRNA and protein were statistically significant in glioma cells compared to blank and negative control groups by detection of real-time PCR and Western blot analysis at72h after transfection with dsP21.2. Since the third days after transfection with dsP21, the proliferation of human glioma cells were significantly depressed.3. The early and late stages of apoptosis were increased in human glioma cells at72h after transfection with dsP21. Analysis of cell cycle distribution revealed that dsP21transfection increased an accumulation in the G0/G1phase and decreased an accumulation in the S phase in human glioma cells. Conclusions:Up-regulation expression of p21by RNAa significantly inhibited cell proliferation, promoted the rates of apoptosis, leaded to G0/G1arrest and remarkably depressed the expression of survivin in human glioma SHG-44cell line. P21activation by RNAa had anti-tumor activity in vitro in human glioma SHG-44cell line. These results suggest that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.
引文
1. Kleihues P, Burger PC, Scheithauer BW. The new WHO classification of brain tumours[J]. Brain Pathol.1993,3(3):255-268.
2. Friese MA, Steinle A, Weller M. The innate immune response in the central nervous system and its role in glioma immune surveillance[J]. Onkologie.2004,27(5):487-491.
3. Bamholtz-Sloan JS, Sloan AE, Schwartz AG. Relative survival rates and patterns of diagnosis analyzed by time period for individuals with primary malignant brain tumor[J]. J Neurosurg. 2003,99(3):458-466.
4. Ohgaki H, Kleihues P. Epidemiology and etiology of gliomas[J]. Acta Neuropathol. 2005,109(1):93-108.
5. Stupp R, Mason WP, van den Bent, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[J]. N Engl J Med.2005,352(10):987-996.
6. DeAngelis LM. Brain tumors[J]. N Engl J Med.2001,344(2):114-123.
7. Reuss D, von DeimLing A. Hereditary tumor syndromes and gliomas[J]. Recent ResuLts Cancer Res.2009,171:83-102.
8. Grigor'ev IuG. The probability of developing brain tumours among users of celluLar telephones (scientific information to the decision of the International Agency for Research on Cancer (IARC) announced on May 31,2011). Radiats Biol Radioecol.2011,51(5):633-638.
9. Michaelis M, Baumgarten P, Mittelbronn M, et al. OncomoduLation by human cytomegalovirus:novel clinical findings open new roads[J]. Med Microbiol Immunol.2011, 200(1):1-5.
10.姚智强,卢亦成.人脑胶质瘤发病分子机制及其临床应用[J].中国肿瘤生物治疗杂志.2008,15(1)：90-94.
11. Louis DN, Ohgaki H, Wiestler OD, et al. The 2007 WHO classification of tumours of the central nervous system[J]. Acta Neuropathol.2007,114(2):97-109.
12. El-Deiry W, Tokino T, VelcuLescu V, et al. WAF1, a potential mediator of p53 tumor suppression[J]. Cell.1993,75(4):817-825.
13. Harper JW, Adami GR, Wei N, et al. The p21 cdk-interacting protein Cipl is a potent inhibitor ofG1 cyclin-dependent kinases[J]. Cell.1993,75(4):805-816.
14. Xiong Y, Hannon G, Zhang H, et al. p21 is a universal inhibitor of cyclin kinases[J]. Nature (Lond.).1993,366(6456):701-704.
15. Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen[J]. Exp. Cell Res.1994,211(1):90-98.
16. Harper JW, Adami GR, Wei N, et al. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases[J]. Cell.1993,75(4):805-816.
17. Polyak K, Kato JY, Solomon MJ, et al. p27Kip1, a cyclin-cdk inhibitor, links transforming growth factor-p and contact inhibition to cell cycle arrest[J]. Genes Dev.1994,8(1):9-22.
18. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin/cdk protein kinase activity, is related to p21[J]. Cell.1994,78(1):67-74.
19. Lee MH, Reynisdottir I, Massague'J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution[J]. Genes Dev.1995,9(6): 639-649.
20. Matsuoka S, Edwards MC, Bai C, et al. p57, a structurally distinct member of the p21 cdk inhibitor family, is a candidate tumor suppressor gene[J]. Genes Dev.1995,9(6):650-662.
21. Polyak K, Lee MH, Erdjument-Bromage H, et al. Cloning of p27Kipl, a cyclin dependent kinase inhibitor and a potential mediator of extracelluLar antimitogenic signals[J]. Cell.1994, 78(1):59-66.
22. Chen J, Jackson PK, Kirschner MW, et al. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA[J]. Nature.1995,374(6520):386-388.
23. Luo Y, Hurwitz J, Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21[J]. Nature.1995,375(6527):159-161.
24. Harper JW, Elledge SJ, Keyomarsi K, et al. Inhibition of cyclin dependent kinases by p21[J]. Mol Biol Cell.1995,6(4):387-400.
25. Zhang H, Xiong Y, Beach D. Proliferating cell nuclear antigen and p21 are components of muLtiple cell cycle kinase complexes[J]. Mol Biol Cell.1993,4(9):897-906.
26. NicuLescu AR, Chen X, Smeets M, et al. Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions:pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication[J]. Mol Cell Biol.1998,18(l):629-643.
27. Ogryzko VV, Wong P, Howard BH. WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases[J]. Mol Cell Biol.1997,17(8):4877-4882.
28.G.克劳斯(著),孙超,刘景生,译.信号转导与调控的生物化学[M].北京：化学工业出版社.2005.
29. Gartel AL, Tyner AL. Transcriptional reguLation of the p21((WAF1/CIP1)) gene[J]. Exp Cell Res.1999,246(2):280-289.
30. Gartel AL, Tyner AL. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis[J]. Mol Cancer Ther.2002,1(8):639-649.
31. Han S, Sidell N, Fisher PB, et al. Up-reguLation of p21 gene expression by peroxisome proliferator-activated receptor gamma in human lung carcinoma cells[J]. Clin Cancer Res. 2004,10(6):1911-1919.
32. Zhang P, Wong C, Liu D, et al. p21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step[J]. Genes Dev.1999,13(2):213-224.
33. Kadowaki Y, Fujiwara T, Fukazawa T, et al. Induction of differentiation-dependent apoptosis in human esophageal squamous cell carcinoma by adenovirus-mediated p21sdil gene transfer[J]. Clin Cancer Res.1999,5(12):4233-4241.
34. Martin-Caballero J, Flores JM, Garcia-Palencia P, et al. Tumor susceptibility of p21(Wafl/Cipl)-deficient mice[J]. Cancer Res.2001,61(16):6234-6238.
35. Topley GI, Okuyama R, Gonzales JG, et al. p21(WAF1/CIp1) functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential[J]. Proc Natl Acad Sci USA.1999,96(16):9089-9094.
36. Poole AJ, Heap D, Carroll RE, et al. Tumor suppressor functions for the Cdk inhibitor p21 in the mouse colon[J]. Oncogene.2004,23(49):8128-8134.
37. Fotedar R, Brickner H, Saadatmandi N, et al. Effect of p21wafl/cipl transgene on radiation induced apoptosis in T cells[J]. Oncogene.1999,18(24):3652-3658.
38. Kondo S, Barna BP, Kondo Y, et al. WAF1/CIP1 increases the susceptibility of p53 non-functional malignant glioma cells to cisplatin-induced apoptosis[J]. Oncogene. 1996,13(6):1279-1285.
39. Lincet H, PouLain L, Remy J S, et al. The p21(cipl/wafl) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells[J]. Cancer Lett. 2000,161(1):17-26.
40. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma[J]. Nat Med.1997,3(8):917-921.
41. Deveraux QL, Reed JC. IAP family proteins:suppressors of apoptosis[J]. Genes Dev. 1999,13(3):239-252.
42. Miller L. An exegesis of IAPs:salvation and surprises from BIR motifs[J]. Trends Cell Biol. 1999,9(8):323-328.
43. Anna P,Genesio L,Andrea P,et al. Expression of Survivin, p53, and caspase3 in Barrett's esophagus carcjnogene5is[J]. Human Pathology.2006,37(1):16-22.
44. Adida C, Crotty PL, McGrath J, et al. Developmentally reguLated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation [J]. Am J Pathol. 1998,152(1):43-49.
45. Kawasaki H, Altieri DC, Lu CD, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer[J]. Cancer Res.1998,58(22):5071-5074.
46. Monzo M, Rosell R, Felip E, et al. A novel anti-apoptosis gene:Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers[J]. J Clin Oncol. 1999,17(7):2100-2104.
47. Kato J, Kuwabara Y, Mitani M, et al. Expression of survivin in esophageal cancer:correlation with the prognosis and response to chemotherapy[J]. Int J Cancer.2001,95(2):92-95.
48.陈涛,贾玉容,赵铁军等survivin反义寡核苷酸对肝癌细胞生长抑制作用的研究[J]. World Chin J Digestol.2004,12(7):1546-1549.
49. Hendruschk S, Wiedemuth R, Aigner A, et al. RNA interference targeting survivin exerts antitumoral effects in vitro and in established glioma xenografts in vivo[J]. Neuro Oncol. 2011,13(10):1074-1089.
50. Mattick JS. Non-coding RNAs:the architects of eukaryotic complexity [J]. EMBO Rep 2001,2(11):986-991.
51. 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.
52. Watanabe T, Totoki Y, Toyoda A, et al. Endogenous siRNA from naturally formed dsRNA reguLate transcripts in mouse oocytes[J]. Nature.2008,453(7194):539-543.
53. Morris KV, Chan SW, Jacobsen SE, et al. Small interfering RNA-induced transcriptional gene silencing in human cells[J]. Science.2004,305(5688):1289-1292.
54. Volpe TA, Kidner C, Hall IM, et al. ReguLation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi[J]. Science.2002,297(5588):1833-1837.
55. Bagga S, Bracht J, Hunter S, et al. ReguLation by let-7 and lin-4 miRNAs resuLts in target mRNA degradation[J]. Cell.2005,122(4):553-563.
56. Li LC, Okino ST, Zhao H, et al. Small dsRNA induce transcriptional activation in human cells[J]. Proc Natl Acad Sci USA.2006,103(46):17337-17342.
57. Janowski BA, Younger ST, Hardy DB, et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs[J]. Nat Chem Biol.2007,3(3):166-173.
58. Portnoy V, Huang V, Place RF, et al. Small RNA and transcriptional upreguLation[J]. Wiley Interdiscip Rev RNA.2011,2(5):748-760.
59. Yue X, Schwartz JC, Chu Y, et al. Transcriptional reguLation by small RNAs at sequences downstream from 3'gene termini[J]. Nat Chem Biol.2010,6(8):621-629.
60. Yin H, Lin H. An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster[J]. Nature.2007,450(7167):304-308.
61. Vasudevan S, Steitz JA. AU-rich-element-mediated upreguLation of translation by FXR1 and Argonaute 2. Cell.2007,128(6):1105-1118.
62. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation:microRNAs can up-reguLate translation[J]. Science.2007,318(5858):1931-1934.
63. Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger:short RNAs that silence gene expression[J]. Nat Rev Mol Cell Biol.2003,4(6):457-467.
64. Place RF, Noonan EJ, Foldes-Papp Z, et al. Defining features and exploring chemical modifications to manipuLate RNAa activity[J]. Curr Pharm Biotechnol.2010,11(5):518-526.
65. Matsui M, Sakurai F, Elbashir S, et al. Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter[J]. Chem Biol. 2010,17(12):1344-1355.
66. Schwartz JC, Younger ST, Nguyen NB, et al. Antisense transcripts are targets for activating small RNAs[J]. Nat Struct Mol Biol.2008,15(8):842-848.
67.陈忠,李龙承RNAa与肿瘤.现代泌尿生殖肿瘤杂科[J].2009,1(1)：1-3.
68. Place RF, Li LC, Pookot D, Noonan EJ, et al. MicroRNA-373 induces expression of genes with complementary promoter sequences[J]. Proc Natl Acad Sci USA.2008, 105(5):1608-1613.
69. Turunen MP, Lehtola T, Heinonen SE, et al. Efficient reguLation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism:a novel example of epigenetherapy[J]. Circ Res.2009,105(6):604-609.
70. Huang V, Qin Y, Wang J, et al. RNAa is conserved in mammalian cells[J]. PLoS ONE.2010, 5(1):e8848.
71. Mao Q, Li Y, Zheng X, et al. Up-reguLation of E-cadherin by small activating RNA inhibits cell invasion and migration in 5637 human bladder cancer cells[J]. Biochem Biophys Res Commun.2008,31,375(4):566-570.
72. Wei Junxia, Gao Ping, Han Yuan, et al. Double strand RNA-guided endogeneous E-cadherin up-reguLation induces the apoptosis and inhibits proliferation of breast carcinoma cells in vitro and in vivo[J]. Cancer Sci.2010,101(8):1790-1796.
73. Chen Z, Place RF, Jia ZJ, et al. Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells[J]. Mol Cancer Ther.2008,7(3):698-703.
74. Wei J, Zhao J, Long M, et al. p21WAFl/CIPl gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell[J]. BMC Cancer.2010,19,10:632.
75. Wu ZM, Dai C, Huang Y, et al. Anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNA in human hepatocelluLar carcinoma cell lines. Acta Pharmacologica Sinica[J]. Acta Pharmacol Sin.2011,32(7):939-946.
76. Whitson JM, Noonan EJ, Pookot D, et al. Double stranded-RNA-mediated activation of P21 gene induced apoptosis and cell cycle arrest in renal cell carcinoma[J]. Int. J. Cancer. Int J Cancer.2009,125(2):446-452.
77. Harada K, Kurisu K, Sadatomo T, et al. Growth inhibition of human glioma cells by transfection-induced P21 and its effects on telomerase activity[J]. J Neurooncol. 2000,47(1):39-46.
78.柳湘,韩俊松,田培坤等.新型基因转移系统介导p2l基因治疗人脑胶质瘤的体外研究[J].中国肿瘤生物治疗杂志.2000,7(1)：11-14.
79. American Cancer Society. Cancer Facts and Figures[J]. Washington, DC:American Cancer Society,2000.
80. Dolecek TA, Propp JM, Stroup NE, et al. CBTRUS statistical report:primary brain and central nervous system tumors diagnosed in the United States in 2005-2009[J]. Neuro Oncol. 2012,14 Suppl 5:vl-49.
81. Riemenschneider MJ, Reifenberger G. Astrocytic tumors[J]. Recent ResuLts Cancer Res. 2009,171:3-24.
82. Mita AC, Mita MM, Nawrocki ST, et al. Survivin:key reguLator of mitosis and apoptosis and novel target for cancer therapeutics[J]. Clin Cancer Res.2008,14(16):5000-5005.
83. Yamamoto H, Ngan CY, Monden M. Cancer cells survive with survivin[J]. Cancer Sci.2008, 99(9):1709-1714.
84. Hoffman WH, Biade S, Zilfou JT, et al. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53[J]. J Biol Chem.2002,277(5):3247-3257.
85. Pennati M, Folini M, Zaffaroni N. Targeting survivin in cancer therapy[J]. Expert Opin Ther Targets.2008,12(4):463-476.
86.甄海宁,章翔,胡佩臻等.生存素基因表达与脑胶质瘤恶性增殖及凋亡关系的研究[J].中华外科杂志.2005,43(13)：885-888.
87. Chakravarti A, Noll E, Black PM, et al. Quantitatively determined survivin expression levels are of prognostic value in human gliomas[J]. J Clin Oncol.2002,20(4):1063-1068
88. Furnari FB, Fenton T, Bachoo RM, et al. Malignant astrocytic glioma:genetics, biology, and paths to treatment[J]. Genes Dev.2007,21(21):2683-2710.
89. Komiya T, Hosono Y, Hirashima T, et al. p21 expression as a predictor for favorable prognosis in squamous cell carcinoma of the lung[J]. Clinical Cancer research.1997,3(10):1831-1835.
90. Y-Z Shi, A-M Hui, T Takayama, et al. Reduced p21WAF1/CIP1protein expression is predominantly related to altered p53 in hepatocelluLar carcinomas[J]. British Journal of Cancer.2000,83(1):50-55.
91.姜又红,隋承光,孟凡东等P21WAF1/CIP1、P16蛋白表达与人脑神经胶质瘤的相关分析[J].肿瘤防治.2005,17(5)：0311-0313.
92. De la Cueva E, Garcia-Cao I, Herranz M, et al. Tumorigenic activity of p21(Waf1/Cip1) in thymic lymphoma[J]. Oncogene.2006,25(29):4128-4132.
93. Shah MA, Kortmansky J, Motwani M, et al. A phase I clinical trial of the sequential combination of irinotecan followed by flavopiridol[J]. Clin Cancer Res.2005, 11(10):3836-3845.
94. Rau B, Sturm I, Lage H, et al. Dynamic expression profile of p21WAF1/CIP1 and Ki-67 predicts survival in rectal carcinoma treated with preoperative radiochemotherapy[J]. J Clin Oncol.2003,21(18):3391-3401.
95. Goan YG, Hsu HK, Chang HC, et al. Deregulated p21(WAF1) overexpression impacts survival of surgically resected esophageal squamous cell carcinoma patients[J]. Ann Thorac Surg.2005,80(3):1007-1016.
96. Adnane J, Jackson RJ, Nicosia SV, et al. Loss of p21 WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/knockout mammary cancer model[J]. Oncogene.2000,19(47):5338-5347.
97. Noseda M, Chang L, McLean G, et al. Notch activation induces endothelial cell cycle arrest and participates in contact inhibition:role of p21Cip1 repression[J]. Mol Cell Biol.2004, 24(20):8813-8822.
98. Gartel AL, Radhakrishnan SK. Lost in transcription:p21 repression, mechanisms, and consequences[J]. Cancer Res.2005,65(10):3980-3985.
99. Gartel AL. The conflicting roles of the cdk inhibitor p21(CIP1/WAF1) in apoptosis[J]. Leuk Res.2005,29(11):1237-1238.
100. Raj K, Ogston P, Beard P. Virus-mediated killing of cells that lack p53 activity[J]. Nature. 2001,412(6850):914-917.
101. Dotto GP. p21(WAF1/Cipl):more than a break to the cell cycle[J]? Biochim Biophys Acta. 2000,1471(1):M43-56.
102. Chang BD, Watanabe K, Broude EV, et al. Effects of p21Waf1/Cip1/Sdi1 on celluLar gene expression:implications for carcinogenesis, senescence, and age-related diseases[J]. Proc Natl Acad Sci USA.2000,97(8):4291-4296.
103. Coqueret O, Gascan H. Functional interaction of STAT3 transcription factor with the cell cycle inhibitor p21 WAF1/CIP1/SDI1 [J]. J Biol Chem.2000,275(25):18794-18800.
104. Fritah A, Saucier C, Mester J, et al. p21WAF1/CIP1 selectively controls the transcriptional activity of estrogen receptor alpha[J]. Mol Cell Biol.2005,25(6):2419-2430.
105. Shen J, Liu J, Long Y, et al. Knockdown of survivin expression by siRNA enhances chemosensitivity of prostate cancer cells and attenuates its tumorigenicity[J]. Acta Biochim Biophys Sin (Shanghai).2009,41(3):223-230.
106. Hou JQ, He J, Wang XL, et al. Effect of small interfering RNA targeting survivin gene on biological behaviour of bladder cancer[J]. Chin Med J (Eng1).2006,119(20):1734-1739.
107. Sasaki T, Lopes MB, Hankins GR, et al. Expression of survivin, an inhibitor of apoptosis protein, in tumors of the nervous system[J]. Acta Neuropathol.2002,104(1):105-109.
108. Jiao B, Yao Z, Geng S, et al. Expression of survivin, a novel apoptosis inhibitor and cell reguLatory protein, in human gliomas[J]. Chin Med J.2004,117:612-614.
109. Chakravarti A, Noll E, Black PM, et al. Quantitatively determined survivin expression levels are of prognostic value in human gliomas[J]. J Clin Oncol.2002,20(4):1063-1068.
110. Xie D, Zeng YX, Wang HJ, et al. Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma[J]. British Journal of Cancer.2006,94(1):108-114.
111. Mahotka C, Liebmann J, Wenzel M, et al. Differential subcelluLar localization of functionally divergent survivin splice variants[J]. Cell Death Differ.2002,9(12):1334-1342
112. Uren AG, Wong L, Pakusch M, et al. Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype[J]. Curr Biol.2000,10(21): 1319-1328.
113. Honda R, Korner R, Nigg EA. Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis[J]. Mol Biol Cell.2003,14(8):3325-3341.
114. Vong QP, Cao K, Li HY, et al. Chromosome alignment and segregation reguLated by ubiquitination of survivin[J]. Science.2005,310(5753):1499-1504.
115. O'Connor DS, Wall NR, Porter AC, et al. A p34(cdc2) survival checkpoint in cancer[J]. Cancer Cell.2002,2(1):43-54.
116. Dohi T, Beltrami E, Wall NR, et al. Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis[J]. J Clin Inves.2004,114(8):1117-1127.
117. Dohi T, Okada K, Xia F, et al. An IAP-IAP complex inhibits apoptosis[J]. J Biol Chem.2004, 279(33):34087-34090.
118. Gratas C, et al. Fas ligand expression in glioblastoma cell lines and primary astrocytic brain tumors[J]. Brain Pathol.1997,7(3):863-869.
119. Miyashita T, et al. Tumor suppressor p53 is a reguLator of bcl-2 and bax gene expression in vitro and in vivo[J]. Oncogene.1994,9(6):1799-1805.
120.王占祥,章翔,费舟等.外源性p21WAF1/CIP1基因转染对人胶质瘤细胞生长和细胞周期的影响[J].第四军医大学学报.2003,24(22)：2030-2033.
121.王文宏,惠国桢,马文雄等.重组腺病毒介导P21 WAF1/CIP1诱导人脑胶质瘤细胞凋亡的体外实验研究[J].东南大学学报(医学版).2002,21(4)：289-292.
122. Hanahan D, Weinberg RA. Hallmarks of cancer:the next generation[J]. Cell.2011, 144(5):646-674.
123. Mao Q, Zheng X, Yang K, et al. Suppression of migration and invasion of PC3 prostate cancer cell line via activating E-cadherin expression by small activating RNA[J]. Cancer Invest.2010,28(10):1013-1018.
124. Wang J, Place RF, Huang V, et al. Prognostic value and function of KLF4 in prostate cancer: RNAa and vector-mediated overexpression identify KLF4 as an inhibitor of tumor cell growth and migration[J]. Cancer Res.2010,70(24):10182-10191.
125. Chen R, Wang T, Rao K, et al. Up-reguLation of VEGF by small activator RNA in human corpus cavernosum smooth muscle cells[J]. J Sex Med.2011,8(10):2773-2780.
126.吴明星,李绍珍,曾骏文等.外源性细胞周期蛋白激酶抑制因子p21基因对人品状体上皮细胞周期的影响[J].中华眼科杂志.2003,39(4)：209-214.
127. Ieda M, Fu JD, Delgado-Olguin P, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors[J]. Cell.2010,142(3):375-386.
128. Szabo E, Rampalli S, Risueno RM, et al. Direct conversion of human fibroblasts to muLtilineage blood progenitors [J]. Nature.2010,468(7323):521-526
129. Vierbuchen T, Ostermeier A, Pang ZP, et al. Direct conversion of broblasts to functional neurons by defined factors[J]. Nature.2010,463(7284):1035-1041.
130. Pecot CV, Calin GA, Coleman RL, et al. RNA interference in the clinic:challenges and future directions[J]. Nat Rev Cance.2011,11(1):59-67.
131. Davis ME, Zuckerman JE, Choi CH, et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[J]. Nature.2010,464(7291):1067-1070.
1 Costa FF. Non-coding RNAs:new players in eukaryotic biology[J]. Gene.2005, 357(2):83-94.
2 Mattick JS. Non-coding RNAs:the architects of eukaryotic complexity [J]. EMBO Rep 2001,2(11):986-991.
3 Andrew Fire, SiQun Xu, Mary K. Montgomery, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
4 Britten RJ, Davidson EH. Science.1969,165(3891):349-57.
5 Li LC, Okino ST, Zhao H, et al. Small dsRNA induce transcriptional activation in human cells[J]. Proc Natl Acad Sci USA.2006,103(46):17337-17342.
6 Janowski BA, Younger ST, Hardy DB, et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs[J]. Nat Chem Biol.2007,3(3):166-173.
7 Huang V, Qin Y, Wang J, et al. RNAa is conserved in mammalian cells[J]. PLoS ONE.2010, 5(1):e8848.
8 Matsui M, Sakurai F, Elbashir S, et al. Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter[J]. Chem Biol. 2010,17(12):1344-1355.
9 Voutila J, S(?)trom P, Mintz P, et al. Gene Expression Profile Changes After Short-activating RNA-mediated Induction of Endogenous Pluripotency Factors in Human Mesenchymal Stem Cells[J]. Mol Ther Nucleic Acids.2012,1:e35.
10 Shibuya K, Fukushima S, Takatsuji H. RNA-directed DNA methylation induces transcriptional activation in plants[J]. Proc Natl Acad Sci USA.2009,106(5):1660-1665.
11 Place RF, Li LC, Pookot D, et al. MicroRNA-373 induces expression of genes with complementary promoter sequences[J]. Proc Natl Acad Sci USA.2008,105(5):1608-1613.
12 Huang V, Place RF, Portnoy V, et al. UpreguLation of Cyclin B1 by miRNA and its implications in cancer[J]. Nucleic Acids Res.2012,40(4):1695-1707.
13 Yue X, Schwartz JC, Chu Y, et al. Transcriptional reguLation by small RNAs at sequences downstream from 3'gene termini[J]. Nat Chem Biol.2010,6(8):621-629.
14 Yin H, Lin H. An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster[J]. Nature.2007,450(7167):304-308.
15 Vasudevan S, Steitz JA. AU-rich-element-mediated upreguLation of translation by FXR1 and Argonaute 2[J]. Cell.2007,128(6):1105-1118.
16 Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation:microRNAs can up-reguLate translation[J]. Science.2007,318(5858):1931-1934.
17 Chu Y, Yue X, Younger ST, et al. Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter[J]. Nucleic Acids Res.2010,38(21):7736-7748.
18 Portnoy V, Huang V, Place RF, et al. Small RNA and transcriptional upreguLation[J]. Wiley Interdiscip Rev RNA.2011,2(5):748-760.
19陈忠,李龙承RNAa与肿瘤.现代泌尿生殖肿瘤杂志[J].2009,1(1)：1-3.
20 Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger:short RNAs that silence gene expression[J]. Nat Rev Mol Cell Biol.2003,4(6):457-467.
21 Janowski BA, Younger ST, Hardy DB, et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs[J]. Nat Chem Biol.2007,3(3):166-173.
22 Turunen MP, Lehtola T, Heinonen SE, et al. Efficient reguLation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism:a novel example of epigenetherapy[J]. Circ Res.2009,105(6):604-609.
23 Hanahan D, Weinberg RA. Hallmarks of cancer:the next generation[J]. Cell.2011, 144(5):646-674.
24 Mao Q, Li Y, Zheng X, et al. Up-reguLation of E-cadherin by small activating RNA inhibits cell invasion and migration in 5637 human bladder cancer cells[J]. Biochem Biophys Res Commun.2008,31,375(4):566-570.
25 Mao Q, Zheng X, Yang K, et al. Suppression of migration and invasion of PC3 prostate cancer cell line via activating E-cadherin expression by small activating RNA[J]. Cancer Invest.2010,28(10):1013-1018.
26 Junxia W, Ping G, Yuan H, et al. Double strand RNA-guided endogeneous E-cadherin up-reguLation induces the apoptosis and inhibits proliferation of breast carcinoma cells in vitro and in vivo[J]. Cancer Sci.2010,101 (8):1790-1796.
27 Chen Z, Place RF, Jia ZJ, et al. Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells[J]. Mol Cancer Ther.2008,7(3):698-703.
28 Wei J, Zhao J, Long M, et al. p21WAFl/CIPl gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell[J]. BMC Cancer.2010,19,10:632.
29 Wu ZM, Dai C, Huang Y, et al. Anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNA in human hepatocelluLar carcinoma cell lines. Acta Pharmacologica Sinica[J]. Acta Pharmacol Sin.2011,32(7):939-946.
30 Whitson JM, Noonan EJ, Pookot D, et al. Double stranded-RNA-mediated activation of P21 gene induced apoptosis and cell cycle arrest in renal cell carcinoma[J]. Int. J. Cancer. Int J Cancer.2009,125(2):446-452.
31 Wang J, Place RF, Huang V, et al. Prognostic value and function of KLF4 in prostate cancer: RNAa and vector-mediated overexpression identify KLF4 as an inhibitor of tumor cell growth and migration[J]. Cancer Res.2010,70(24):10182-10191.
32 Ieda M, Fu JD, Delgado-Olguin P, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors[J]. Cell.2010,142(3):375-386.
33 Szabo E, Rampalli S, Risueno RM, et al. Direct conversion of human fibroblasts to muLtilineage blood progenitors[J]. Nature.2010,468(7323):521-526
34 Vierbuchen T, Ostermeier A, Pang ZP, et al. Direct conversion of broblasts to functional neurons by defined factors[J]. Nature.2010,463(7284):1035-1041.
35 Pecot CV, Calin GA, Coleman RL, et al. RNA interference in the clinic:challenges and future directions[J]. Nat Rev Cance.2011,11(1):59-67.
36 Davis ME, Zuckerman JE, Choi CH, et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[J]. Nature.2010,464(7291):1067-1070.