摘要
Gankyrin是近年来在肝细胞癌中发现的一个新的致癌基因编码的蛋白,因其几乎在所有的肝癌患者的癌组织中均存在过表达而受到重视。Gankyrin蛋白可以与p16~(INK4A)竞争结合Cdk4,从而对抗p16~(INK4A)对Cdk4的抑制作用,进而对抗了p16~(INK4A)对肿瘤细胞增殖的抑制作用。Gankyrin的表达升高可以引起Rb磷酸化水平上升,导致转录因子E2F1的活性释放,引起细胞增殖的一系列相关基因的转录激活。此外由于gankyrin蛋白存在于26s蛋白酶体的19s调节复合体中,且可与Mdm2发生相互作用,而Mdm2属于泛肽连接酶,介导泛素/26s蛋白酶体对P53的降解,因此认为gankyrin可能参与了P53的降解过程。p16~(INK4A)、Rb、P53等的失活目前被认为是肝癌发生过程中最为关键的几个因素,而gankyrin则同时参与在三个过程之中。近来有实验在Fischer 344鼠体内诱发肝癌,结果发现在肿瘤发生的最初阶段,即出现了gankyrin的表达升高,这些均提示我们gankyrin可能是肝癌发生发展过程中的一个关键基因,同时也为肝癌的治疗提供了一个潜在的靶
Gankyrin is a novel gene identified from human hepatocellular carcinoma,which is overexpressed in almost all studied hepatocellular carcinomas. Gankyrin binds to Cdk4 and counteracts the inhibitory function of the tumor suppressors p16~INK4A. Overexpression of gankyrin increases both the phosphorylation and degradation of Rb.And inactivation of RBI by phosphorylation seems to release E2F from an inhibitory complex, enabling it to promote the transcription of genes necessary for progression into late G1 and S phases.Because gankyrin protein is one of the non-ATPase subunits of 19 S complex,which is a 700 kDa multisubunit regulatory complex of the human 26 S proteasome,and gankyrin can interact with Mdm2 ,which is an E3 ubiquitin ligase involved in p53 degradation, So gankyrin is thought to be related with the degradation of p53.It was reported that the inactivation of some tumor suppressive genes such as p53 ,pl6~INK4Aand Rb is critical to tumorigenesis in HCCs,and gankyrin seems to be involved in all three progression.Recently,researchers induced hepatocellular carcinoma in Fisher 344 rats and observed which genes involved in the tumorigenetic progress,the results showed that gankyrin is overexpressed from the earliest stage of tumor development. These findings suggest that gankyrin is a major player in cell
cycle control and tumorigenesis in HCCs.lt also provided us a promising target for therapy.The present study consists of three parts:Part One: The expression of gankyrin gene in hepatocellular carcinoma cells and the gene's clone, prokaryotic expression and antibody preparationAIM: To determine the expression of gankyrin gene in hepatocellular carcinoma cell lines . To express gankyrin fusion protein in E.coli and to prepare antibody against it. METHODS:The expression of gankyrin in HepG2,SMMC-7721 and HHCC cell lines was detected using RT-PCR analysis. The core fragment of gankyrin was cloned into plasmid pGEX-4T-2 containing glutathione s-transferase(GST) fusion protein gene and plasmid pRSET-B containing His fusion protein.Following restriction enzyme digestion analysis and sequencing.Reformed plasmid was transformed into E.coli DH-5a. Gankyrin fusion protein was expressed under IPTG induction .Then the purified gankyrin protein was used to immunize New Zealand rabbits.The specific antibody against Gankyrin was identified by ELISA and Western blot. RESULTS: The expression of gankyrin mRNA and protein could be detected in all three hepatocellular carcinoma cell lines. pGEX-4T-Gankyrin and pRSET-B-Gankyrin was constructed successfully,but only GST-Gankyrin fusion protein was expressed in E.coli and it specific polyclonal antibody was obtained. CONCLUTION: Gankyrin gene expressed in HepG2,SMMC-7721,HHCC cell lines. The successful expression of GST-Gankyrin fusion protein in E.coli and the preparation of it's specific polyclonal antibody will be valuable for the study of the biological function of gankyrin.Part Two: Suppressing the expression of gankyrin in human hepatocellular carcinoma cell by RNAiAIM: To construct the plasmid containing short hairpin RNA (shRNA)
targeting at gankyrin in order to inhibit the expression of gankyrin in HepG2 cell lines. METHODS: Three couple of shRNA sequence corresponding to gankyrin were synthesized and inserted into plasmid pGenesil-1 under U6 promoter to construct recombinant plasmid pGenesil-1 -Gankyrin 1 ^ pGenesil-1-GankyriiL^ pGenesil-l-Gankyrin3, and the vector expressing shRNA which is irrelevant with all human gene was used as control (pGenesil-1- control) ,then HepG2 cell lines stably transfected with them were established and gene expression inhibition was assessed by RT-PCR and Western blotting analysis. RESULTS: The recombinant plasmid pGenesil-1-Gankyrin 1 > pGenesil-1-Gankyrh^ pGenesil-l-Gankyrin3 and pGenesil-1- control were successfully constructed. PGenesil-1-Gankyrin2> pGenesil-l-Gankyrin3 suppress the expression of gankyrin significantly in HepG2. CONCLUTION: The result showed the shRNA of gankyrin can efficiently inhibited the expression of gankyrin in HepG2. RNAi mediated by shRNA is a useful methods in gankyirn gene silence.Part Three: Effects on proliferation of suppressing the expression of gankyrin in human hepatocellular carcinoma cell line HepG2AIM: To explore the effect on morphology,proliferation , cell cycle and tumor formation in nude mice when suppressing gankyrin expression in HepG2 cell line. METHODS: Observe the cells stably transfected with different plasmid under electron microscope.The inhibitory effect on cell growth was studied by cell counting and MTT assay, and cell cycle was analyzed by flow cytometry. Then tumor formation experiment was carried on nude mice.RESULTS: Compared with the cells transfected with control, electron microscopy show the cell has lower ratio between nuclear and plasma,smaller nucleolus and lower activity when suppressing the expression of gankyrin.And the growth of HepG2 cell was inhibited significantly , the number of the cells was much lower,MTT assay showed cell proliferation index decreased,flow cytometric analysis demonstrated that the cell cycle was stucked at Gl phase. Suppressing gankyrin also inhibited tumor formation in
引文
1 Sangro B, Mazzollini G, Prieto J.Future therapies for hepatocellular carcinoma. Eur J Gastroenterol Hepatol. 2005 May; 17(5):515-21.
2 Nita ME, Alves VA, Carrilho FJ, Ono-Nita SK, Mello ES, Gama-Rodrigues JJ.Molecular aspects of hepatic carcinogenesis.Rev Inst Med Trop Sao Paulo. 2002 Jan-Feb;44(1):39-48.
3 Higashitsuji H, Itoh K, Nagao T, Dawson S, Nonoguchi K, Kido T, Mayer RJ, Arii S, Fujita J. Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas. Nat Med 2000;6:96-9.
4 Fu XY, Wang HY, Tan L, Liu SQ, Cao HF, Wu MC. Overexpression of p28/gankyrin in human hepatocellular carcinoma and its clinical significance. World J Gastroenterol 2002;8:638-43.
5 Marotta F, Vangieri B, Cecere A, Gattoni A.The pathogenesis of hepatocellular carcinoma is multifactorial event. Novel immunological treatment in prospect.Clin Ter. 2004 May; 155(5): 187-99.
6 Feitelson MA, Pan J, Lian Z.Early molecular and genetic determinants of primary liver malignancy.Liver Int. 2003 Dec;23(6):405-9.
7 Kew MC.Synergistic interaction between aflatoxin B1 and hepatitis B virus in hepatocarcinogenesis.Liver Int. 2003 Dec;23(6):405-9.
8 Tang ZY, Ye SL, Liu YK, Qin LX, Sun HC, Ye QH, Wang L, Zhou J, Qiu S J, Li Y, Ji XN, Liu H, Xia JL, Wu ZQ, Fan J, Ma ZC, Zhou XD, Lin ZY, Liu KD.A decade's studies on metastasis of hepatocellular carcinoma.J Cancer Res Clin Oncol. 2004 Apr; 130(4): 187-96.
9 Staib F, Hussain SP, Hofseth LJ, Wang XW, Harris CC.TP53 and liver carcinogenesis.Hum Mutat. 2003 Mar;21 (3):201-16.
10 Tannapfel A, Wittekind C.Genes involved in hepatocellular carcinoma: deregulation in cell cycling and apoptosis.Virchows Arch. 2002 Apr;440(4):345-52.
11 Matsumoto Y, Fujii H, Matsuda M, Kono H.Multicentric occurrence of hepatocellular carcinoma: diagnosis and clinical significance.J Hepatobiliary Pancreat Surg. 2001;8(5):435-40.
12 Qin Y, Liu JY, Li B, Sun ZL, Sun ZF.Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis.World J Gastroenterol. 2004 May 1;10(9):1276-80.
13 Qin Y, Liu JY, Li B, Peng WZ, Fu MD, Sun ZL, Sun ZF.Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis.Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004 Apr;21(2): 132-7.
14 Wang ZW, Peng ZH, Tang HM, Qiu GQ, Xing TH, Yang MZ.The effects of pl6 and Rb protein expression and NF-kappaB activity on hepatocarcinogenesis.Zhonghua Yi Xue Za Zhi. 2004 Feb 2;84(3):225-8.
15 Narimatsu T, Tamori A, Koh N, Kubo S, Hirohashi K, Yano Y, Arakawa T, Otani S, Nishiguchi S.p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection.Intervirology. 2004;47(1):26-31.
16 Matsuda Y, Ichida T, Genda T, Yamagiwa S, Aoyagi Y, Asakura H.Loss of pl6 contributes to p27 sequestration by cyclin D(1)-cyclin-dependent kinase 4 complexes and poor prognosis in hepatocellular carcinoma.Clin Cancer Res. 2003 Aug 15;9(9):3389-96.
17 Cho JW, Jeong YW, Han SW, Park JB, Jang BC, Baek WK, Kwon TK, Park JW, Kim SP, Suh MH, Suh SI.Aberrant p16INK4A RNA transcripts expressed in hepatocellular carcinoma cell lines regulate pRb phosphorylation by binding with CDK4, resulting in delayed cell cycle progressionXiver Int. 2003 Jun;23(3):194-200.
18 Edamoto Y, Hara A, Biernat W, Terracciano L, Cathomas G, Riehle HM, Matsuda M, Fujii H, Scoazec JY, Ohgaki H.Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis.Int J Cancer. 2003 Sep 1;106(3):334-41.
19 Shim YH, Yoon GS, Choi HJ, Chung YH, Yu E.p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis.Cancer Lett. 2003 Feb 20;190(2):213-9.
20 Yuan C, Li J, Mahajan A, Poi MJ, Byeon IJ, Tsai MD. Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure-function relationship. Biochemistry 2004;43:12152-61.
21 Dawson S, Apcher S, Mee M, Higashitsuji H, Baker R, Uhle S, Dubiel W, Fujita J, Mayer RJ.Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome.J Biol Chem. 2002 Mar 29;277(13): 10893-902.
22 Krzywda S, Brzozowski AM, Al-Safty R, Welchman R, Mee M, Dawson S, Fujita J, Higashitsuji H, Mayer RJ, Wilkinson AJ.Crystallization of gankyrin, an oncoprotein that interacts with CDK4 and the S6b (rpt3) ATPase of the 19S regulator of the 26S proteasome.Acta Crystallogr D Biol Crystallogr. 2003 Jul;59(Pt 7): 1294-5.
23 Krzywda S, Brzozowski AM, Higashitsuji H, Fujita J, Welchman R, Dawson S, Mayer RJ, Wilkinson AJ.The crystal structure of gankyrin, an oncoprotein found in complexes with cyclin-dependent kinase 4, a 19 S proteasomal ATPase regulator, and the tumor suppressors Rb and p53.J Biol Chem. 2004 Jan 9;279(2):1541-5.
24 Padmanabhan B, Adachi N, Kataoka K, Horikoshi M.Crystal structure of the homolog of the oncoprotein gankyrin, an interactor of Rb and CDK4/6.J Biol Chem. 2004 Jan 9;279(2):1546-52.
25 Manjasetty BA, Quedenau C, Sievert V, Bussow K, Niesen F, Delbruck H, Heinemann U.X-ray structure of human gankyrin, the product of a gene linked to hepatocellular carcinoma.Proteins. 2004 Apr 1;55(1):214-7.
26 杜海宁,胡红雨.锚蛋白重复序列介导的蛋白质-蛋白质相互作用.生物化学与生物物理进展.2002;29(1):6-9.
27 Breeden L, Nasmyth K.Similarity between cell-cycle genes of budding yeast and fission yeast and the Notch gene of Drosophila.Nature. 1987 Oct 15-21 ;329(6140):651-4.
28 Breeden L, Nasmyth K.Cell cycle control of the yeast HO gene: cis-and trans-acting regulators.Cell. 1987 Feb 13;48(3):389-97.
29 Sedgwick SG, Smerdon SJ.The ankyrin repeat: a diversity of interactions on a common structural framework.Trends Biochem Sci. 1999 Aug;24(8):311-6.
30 Foord R, Taylor IA, Sedgwick SG, Smerdon SJ.X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function.Nat Struct Biol. 1999 Feb;6(2):157-65.
31 Gorina S, Pavletich NP.Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2.Science. 1996 Nov 8;274(5289):1001-5.
32 Shimada M, Satoh N, Yokosawa H.Involvement of Rel/NF-kappaB in regulation of ascidian notochord formation.Dev Growth Differ. 2001 Apr;43(2): 145-54.
33 Foord R, Taylor IA, Sedgwick SG, Smerdon SJ.X-ray structural analysis of the yeast cell cycle regulator Swi6 reveals variations of the ankyrin fold and has implications for Swi6 function.Nat Struct Biol. 1999 Feb;6(2):157-65.
34 Yamakuni T, Yamamoto T, Hoshino M, Song SY, Yamamoto H, Kunikata-Sumitomo M, Minegishi A, Kubota M, Ito M, Konishi S.A novel protein containing Cdc10/SWI6 motifs regulates expression of mRNA encoding catecholamine biosynthesizing enzymes.J Biol Chem. 1998 Oct 16;273(42):27051-4.
35 von Bohlen und Halbach O, Schober A, Krieglstein K.Genes, proteins, and neurotoxins involved in Parkinson's disease.Prog Neurobiol. 2004 Jun;73(3):151-77.
36 Hori T, Kato S, Saeki M, DeMartino GN, Slaughter CA, Takeuchi J, Toh-e A, Tanaka K. cDNA cloning and functional analysis of p28 (Nas6p) and p40.5 (Nas7p), two novel regulatory subunits of the 26S proteasome.Gene. 1998 Aug 17;216(1):113-22.
37 Fu X, Tan L, Liu S, Li H, Chen L, Qin J, Wu M, Wang H.A novel diagnostic marker, p28GANK distinguishes hepatocellular carcinoma from potential mimics. J Cancer Res Clin Oncol. 2004 Sep;130(9):514-20.
38 Park TJ, Kim HS, Byun KH, Jang JJ, Lee YS, Lim IK. Sequential changes in hepatocarcinogenesis induced by diethylnitrosamine plus thioacetamide in Fischer 344 rats: induction of gankyrin expression in liver fibrosis, pRB degradation in cirrhosis, and methylation of p16(INK4A) exon lin hepatocellular carcinoma. Mol Carcinog 2001;30:138-50.
39 Lim IK. Spectrum of molecular changes during hepatocarcinogenesis induced by DEN and other chemicals in Fisher 344 male rats. Mech Ageing Dev 2003;124:697-708.
40 Iwai A, Marusawa H, Kiuchi T, Higashitsuji H, Tanaka K, Fujita J, Chiba T. Role of a novel oncogenic protein, gankyrin, in hepatocyte proliferation. J Gastroenterol 2003;38: 751-8.
41 Qin JM, Fu XY, Li SJ, Liu SQ, Zeng JZ, Qiu XH, Wu MC, Wang HY. Gene and protein expressions of p28/GANK in rat with liver regeneration. World J Gastroenterol 2003;9:2523-7.
42 Johnson DG, Schneider-Broussard R.Role of E2F in cell cycle control and cancer.Front Biosci. 1998 Apr 27;3:d447-8.
43 Pierce AM, Schneider-Broussard R, Philhower JL, Johnson DG.Differential activities of E2F family members: unique functions in regulating transcription.Mol Carcinog. 1998 Jul;22(3):190-8.
44 Li J, Tsai MD.Novel insights into the INK4-CDK4/6-Rb pathway: counter action of gankyrin against INK4 proteins regulates the CDK4-mediated phosphorylation of Rb.Biochemistry. 2002 Mar 26;41(12):3977-83.
45 Spataro V, Norbury C, Harris AL.The ubiquitin-proteasome pathway in cancer.Br J Cancer. 1998;77(3):448-55.
46 Zavrski I, Jakob C, Schmid P, Krebbel H, Kaiser M, Fleissner C, Rosche M, Possinger K, Sezer O.Proteasome: an emerging target for cancer therapy.Anticancer Drugs. 2005 Jun;16(5):475-81.
47 Berezutskaya E, Bagchi S.The human papillomavirus E7 oncoprotein functionally interacts with the S4 subunit of the 26 S proteasome.J Biol Chem. 1997 Nov 28;272(48):30135-40.
48 Rezvani K, Mee M, Dawson S, McIlhinney J, Fujita J, Mayer RJ.Proteasomal interactors control activities as diverse as the cell cycle and glutaminergic neurotransmission.Biochem Soc Trans. 2003 Apr;31(2):470-3.
49 French SW, Mayer RJ, Bardag-Gorce F, Ingelman-Sundberg M, Rouach H, Neve And E, Higashitsuji H.The ubiquitin-proteasome 26s pathway in liver cell protein turnover: effect of ethanol and drugs.Alcohol Clin Exp Res. 2001 May;25(5 Suppl ISBRA):225S-229S.
50 Blagosklonny MV.Do VHL and HIF-1 mirror p53 and Mdm-2? Degradation-transactivation loops of oncoproteins and tumor suppressors.Oncogene. 2001 Jan 18;20(3):395-8.
51 Nagao T, Higashitsuji H, Nonoguchi K, Sakurai T, Dawson S, Mayer RJ, Itoh K, Fujita J. MAGE-A4 interacts with the liver oncoprotein gankyrin
and suppresses its tumorigenic activity. J Biol Chem 2003;278:10668-74.
52 Izant J , Weintraub H. Inhibition of thymidine kinase gene expression by anti- sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007-15.
53 Jorgensen R. Altered gene expression in plants due to trans in teractions between homologous genes. Trends Biotechnol ,1990 ,8 :340-344.
54 Wassenegger M. Gene silencing. Int Rev Cytol, 2002 ,219:61-113.
55 Cogoni C ,Macino G. Post - transcriptional gene silencing across kingdoms . Genes Dev ,2000,10 : 638-643.
56 Hammond SM , Caudy AA , Hannon GJ . Post - transcriptional gene silencing by double - stranded RNA . Nature Rev Gen , 2001,2 : 110-119.
57 Tabara H , Grishok A , Mello C C. RNAi in C. elegans soaking in the genome sequence. Science , 1998,282 : 430~431.
58 Lin SL , Chuong CM, Ying SY. A novel mRNA - cDNA interference phenomenon for silencing bcl - 2 expression in human LNCaP cells. Biochem Biophys Res Commun, 2001 ,281(3) :639-44.
59 Fire A, Xu S , Montgomery M K, Kostas S A, Driver S E , Mello C C ,. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans . Nature, 1998,391 : 806-811.
60 Pal-Bhadra M, Bhadra U, Birchler J A. Cosuppression of nonhomologous transgenes in Drosophila involves nutuallyu related endogenous sequences. Cell, 1999,99 : 35-46
61 Sanchez-Alvarado A ,Newmark P A. Double- stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci USA, 1999,96: 504-505.
62 Bernstein E , Denli AM, Hannon GJ . The rest is silence. RNA , 2001 ,7 (11):1509-1521
63 Yang S , Tutton S , Pierce E , Yoon K. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol,2001,21(22):7807-7816
64 Hamilton AJ , Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants . Science ,1999 ,286 : 950-952.
65 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi : double - stranded RNA directs the ATP - dependent cleavage of mRNA at21 to 23 nucleotide intervals . Cell ,2000 ,101:25-33.
66 Nykanen A , Haley B , Zamore P D. ATP requirements and small inter ferings RNA structure in the RNA interference pathway, cell, 2001 ,107 (3):309-321.
67 McManus MT, Haines BB, Dillon CP, Whitehurst CE, van Parijs L, Chen J, Sharp PA. Small interfering RNA mediated gene silencing in T lymphocytes . J Immunol ,2002 ,169 (10) :5754-5760.
68 Hutvagner G, Zamore PD. RNAi : nature abhors a double - strand . Curr Opin Genet Dev. 2002 Apr;12(2):225-32.
69 Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K.Identification of essential genesin cultured mammalian cells using small interfering RNAs. J Cell Science ,2001 ,114(24) :4557-65.
70 Bernstein E , Caudy A A , Hammond SM, Hannon GJ. Role for a bidentate rebonuclease in the initiation step of RNA entergerence. Nature,, 2001,409:363-366
71 Hammond S M, Bernstern E , Beach D , Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature , 2000,404 : 293~296
72 Nykanen A , Haley B , Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell ,2001 ,107 :309-321.
73 Hannon GY.RNA interference . Nature ,2002 ,418 :244-251.
74 Lipardi C , Wei Q , Paterson B M. RNAi as random degradative PCR.siRNA primers convert mRNA into dsRNAs that are degraded to generatenew siRNAs. Cell, 2001,107(3) :297-307.
75 Lipardi C, Wei Q, Paterson BM.RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.Cell. 2001 Nov 2;107(3):297-307.
76 Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A.On the role of RNA amplification in dsRNA-triggered gene silencing.Cell. 2001 Nov 16;107(4):465-76.
77 Elbashir SM , Harborth J , Lendeckel W, Yalcin A , Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells . Nature, 2001 ;411 (6836) :494 - 498.
78 Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.Nature Biotechnology, 2002 , 20(5) :497-500.
79 Brummelkamp TR , Bernards R , Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science , 2002 ,296 :550-553.
80 Zamore P D. RNA interference : listening to the sound of silence. Nature Struct Biol, 2001,8 (9) :746-750.
81 Wilda M, Fuchs U , Wossmann W, Borkhardt A. Killing of leukemic cells with aBCR/ ABL fusion gene by RNA interference (RNAi). Oncogene , 2002 ,21(37) :5716-5724.
82 Oh WJ, Kim EK, Ko JH, Yoo SH, Hahn SH, Yoo OJ.Nuclear proteins that bind to metal response element a (MREa) in the Wilson disease gene promoter are Ku autoantigens and the Ku-80 subunit is necessary for basal transcription of the WD gene.Eur J Biochem. 2002 Apr;269(8):2151-61.
83 McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA.RNA interference in adult mice.Nature. 2002 Jul 4;418(6893):38-9.
84 Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A, Salvaterra P, Rossi J.Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.Nat Biotechnol. 2002 May;20(5):500-5.
85 Lindenbach BD,Rice CM. RNAi targeting an animal virus:news from the front. Mol Cell ,2002,9 (5) :925-927.
86 Wilda M, Fuchs U, Wossmann W, Borkhardt A.Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene. 2002 Aug 22;21 (37):5716-24.
87 Sui G, Soohoo C, Affarel B, Gay F, Shi Y, Forrester WC, Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99:5515-5520.
88 Mweda I, Kohara Y, Yamamoto M,Sugimoto A. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi.Curr Biol, 2001,11(3):171-176.
89 李春英,高天文,刘玉峰.酪氨酸酶相关蛋白21的基因克隆表达及纯化.中国皮肤性病学杂志.2001;15(6);368-70.
90 Peng JR, Chen HS, Mou DC, Cao J, Cong X, Qin LL, Wei L, Leng XS, Wang Y, Chen WF.Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters.Cancer Lett. 2005 Mar 10;219(2):223-32.
91 Yang SZ, Dong JH, Zhu J, Li K, Zhang Y.Detection of alpha-fetoproteins mRNA and melanoma agen-1 mRNA in periphera blood of patients with hepatocellular carcinoma and its clinical significance.Zhonghua Wai Ke Za Zhi. 2004 Sep 7;42(17):1060-3.
92 Zhao L, Mou DC, Leng XS, Peng JR, Wang WX, Huang.L, Li S, Zhu JY.Expression of cancer-testis antigens in hepatocellular carcinoma.World J Gastroenterol. 2004 Jul 15; 10(14):2034-8.
93 晏伟,王文亮,张萍,等.一个新的人信号识别微粒受体基因APMCF1的克隆、表达和抗体制备.第四军医大学学报.2003:24(11):964-967.
94 岳乔红,于文彬,郝晓柯,等.人羧肽酶A1活性中心基因的克隆及原核表达.第四军医大学学报.2003:24(23):2132-2135.
95 张爱莲,赵干,王宾,张富春.草原兔尾鼠GST-LZP3融合蛋白的原核表达及其抗体制备.细胞与分子免疫学杂志.2004,20(2):168-170.
96 莫永炎,刘亚伟,王静珍,邓鹏,秦清和,杨志刚,姜勇.人GST-AWPI融合蛋白的原核表达及其抗体制备.中国生物化学与分子生物学报.2003,19(2):168-172.
97 方琴,孙等军,张旭,任志娟,肖芸,刘丰丰,吴正宇,文小平,王季石.TRAIL基因原核表达载体的构建、表达与抗体制备.中国肿瘤生物治疗杂志.2004;11(1):50～53.
98 Yuan B, Latek R, Hossbach M, Tuschl T, Lewitter F.siRNA Selection Server: an automated siRNA oligonucleotide prediction server.Nucleic Acids Res. 2004 Jul 1;32:W 130-4.
99 Balla T, Varnai P.Visualizing cellular phosphoinositide pools with GFP-fused protein-modules.Sci STKE. 2002 Mar 26;2002(125):PL3.
100 Nagy P, Arndt-Jovin DJ, Jovin TM. Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbBl-overexpressing cells. Exp Cell Res 2003; 285:39-49.
101 Timpson P, Lynch DK, Schramek D, Walker F, Daly RJ.Cortactin overexpression inhibits ligand-induced down-regulation of the epidermal growth factor receptor.Cancer Res. 2005 Apr 15;65(8):3273-80.
102 Attwooll C, Denchi EL, Helin K.The E2F family: specific functions and overlapping interests.EMBO J. 2004 Dec 8;23(24):4709-16.
103 Bracken AP, Ciro M, Cocito A, Helin K.E2F target genes: unraveling the biology.Trends Biochem Sci. 2004 Aug;29(8):409-17.
104 Yamasaki L, Pagano M.Cell cycle, proteolysis and cancer.Curr Opin Cell Biol.2004 Dec;16(6):623-8.
105 Frolov MV, Dyson NJ.Molecular mechanisms of E2F-dependent activation and pRB-mediated repression.J Cell Sci. 2004 May 1;117(Pt 11):2173-81.
106 Gerolami R, Uch R, Brechot C, Mannoni P, Bagnis C.Gene therapy of hepatocarcinoma: a long way from the concept to the therapeutical impact.Cancer Gene Ther. 2003 Sep;10(9):649-60.
107 Iimuro Y, Fujimoto J.Strategy of gene therapy for liver cirrohosis and hepatocellular carcinomaJ Hepatobiliary Pancreat Surg. 2003;10(l):45-7.
108 Havlik R, Kral V, Habib N.Gene therapy of liver tumors: results of the first clinical studies.Cas Lek Cesk. 2003;142(9):528-9.
109 Stefani AL, Barzon L, Castagliuolo I, Guido M, Pacenti M, Parolin C, Farinati F, Palu G.Systemic efficacy of combined suicide/cytokine gene therapy in a murine model of hepatocellular carcinomaJ Hepatol. 2005 May;42(5):728-35.
110 Yang DF, Zhu HF, Shen GX, Tian DY.The construction of transferrin receptor- mediated HSV-TK gene transfer system and its effect on human hepatocellular carcinoma cells in vitro.Zhonghua Gan Zang Bing ZaZhi.2004 Feb;12(2):88-91.
111 Sun XY, Wu ZD, Hu JB.Suicide gene therapy of hepatocellular carcinoma and delivery procedure and route of therapeutic gene in vivo.Hepatobiliary Pancreat Dis Int. 2002 Aug;1(3):373-7.
112 Harada N, Shimada M, Okano S, Suehiro T, Soejima Y, Tomita Y, Maehara Y.IL-12 gene therapy is an effective therapeutic strategy for hepatocellular carcinoma in immunosuppressed mice.J Immunol. 2004 Dec 1;173(11):6635-44.
113 Hisaka T, Yano H, Ogasawara S, Momosaki S, Nishida N, Takemoto Y,