高效表达RKIP基因对人喉鳞癌化疗增敏作用的实验研究
|
摘要
探讨高效表达RKIP基因联合化疗药物对人喉鳞癌细胞的作用,为喉癌基因联合化疗减低喉癌化疗抵抗提供了理论依据。应用DNA克隆技术构建干涉和高效表达RKIP的重组质粒,并进行测序鉴定和效果鉴定;应用真核细胞转染技术将重组质粒转染人喉癌Hep-2细胞,观察RKIP表达水平变化对喉鳞癌细胞增殖、周期、凋亡及侵袭的影响,并分析其作用机制;高效表达RKIP的重组质粒联合顺铂后,探讨其减低喉癌化疗抵抗和增强喉癌对顺铂敏感性的可能性。(1)经酶切和测序证实,成功构建重组质粒。荧光定量RT-PCR和Western印迹检测RKIP mRNA和蛋白的表达,高效表达组升高,干涉组表达降低,其中干涉组的RKIP-shRNA501抑制作用最为明显。(2)MTT检测结果显示,高效表达组出现增殖抑制,干涉组出现增殖促进。FCM检测结果显示,高效表达组的G1期细胞增多,出现明显的凋亡峰。干涉组的G1期细胞减少,未出现明显的凋亡峰。Matrigel侵袭实验结果显示,高效表达组细胞侵袭力减弱,干涉组细胞侵袭力增强。MAPK通路的改变或许是RKIP引起喉鳞癌Hep-2细胞增殖、周期、凋亡、侵袭等改变的原因或部分原因。(3)高效表达RKIP的重组质粒联合顺铂后,MTT检测结果显示,细胞增殖受到明显抑制。FCM检测结果显示,细胞凋亡明显增强,MAPK通路的改变或许是RKIP-pIRES2-EGFP增强DDP对喉鳞癌Hep-2细胞影响的原因或部分原因。高效表达RKIP基因联合化疗对人喉鳞癌具有显著的治疗效果。
Head and neck squamous cell carcimoma (HNSCC) is a common malignant tumor in human body, which exhibits rapid growth, strong invasiveness and metastasis and has a high rate of recurrence. Carcinogenesis is a complicate staging process. From the point of view in molecular biology, major pathogenesy is reinforcement of oncogene and metastasis gene activity or inactivation of anti-oncogene and metastasis suppressor gene in human body interacting with various extent and internal factors. Combined radiotherapy and chemotherapy with operation has become a chief method and tendency for this disease. But management of HNSCC is a puzzled question in tumor therapy right along for its low sensitivity and strong resistance to chemotherapy. Meanwhile, the side effect and injury to normal tissue caused by chemotherapy can not be ignored. Therefore, it is urgent to find chemo sensitizer to enhance tumor chemo-sensitization and decrease injury in normal tissues. Due to the advantages of gene therapy, which include uneasy to produce drug resistance and strong specificity, it offers hope in this regard. Gene therapy involves the introduction of gene DNA into cells to exert effects by vector, which will selectively kill cancer cells with no toxicity to the surrounding non-malignant cells. The key point of gene therapy is the choice of target. Through such a process we can study and choose effective, low toxic cure medicine and method for oncotherapy, it is a new direction of oncotherapy to combine orthodox chemotherapy with gene therapy.
Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine binding protein family, a ubiquitously expressed and evolutionarily conserved group of proteins. In recent years, it has been reported that RKIP influence intracellular signaling cascades, cell cycle regulation, the suppression of metastasis, neuro generative processes, the modulation of emotions, and reproduction, and RKIP has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors and enhance tumor chemo-sensitization, but little is known about the role and the regulating mechanisms of RKIP in laryngocarcinoma.
RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing, resulting in the corresponding gene deletion phenotype. It is a hot spot in functional genomics investigation which develops gradually to become a critical technique for defining gene function.
In this study, we transfected recombinant plasmid to Hep-2 cell line with liposome to investigate the therapeutic value and possible mechanism of laryngeal carcinoma by regulating RKIP combined with DDP. Therefore, a theoretical basis and new utilization may be provided on enhancing combined therapeutic efficacy and improving prognosis of head and neck malignant tumors.
Objective: (1) To construct the recombinant plasmid of RKIP short hairpin RNA and high efficiency expression and to verify their efficacy. (2) To observe the effect of RKIP expression levels on cell proliferation, cell cycle, apoptosis, invasiveness and migration in Hep-2 cells, and investigate generation pathogenesy. (3) To observe the possibility of reducing laryngocarcinoma chemotherapy resistance and enhancing laryngocarcinoma sensitivity to DDP after high efficiency expression RKIP combined with DDP demonstrated by the hyperplasia and apoptosis of laryngeal squamous carcinoma cell, and investigate generation pathogenesy.
Method: (1) RKIP short hairpin RNA and high efficiency expression vector were constructed by molecular cloning technique and identified by enzyme digestion and sequencing. After transfection of Hep-2 cells with recombinant plasmid, transfection efficiency was observed and deteced by fluorescence microscope and FCM. Quantitative reverse transcriptase-polymerase chain and Western blotting were used to detect RKIP mRNA and protein expression. (2) After transfection of Hep-2 cells with recombinant plasmid, cell proliferation was determined using MTT method. Changes of cell cycle and apoptotic rates were measured by flow cytometry. Cell invasiveness was evaluated by Matrigel invasion assay. Western blot detected the effects of different RKIP levels upon MAPK pathway. (3) After transfection of Hep-2 cells with high efficiency expression vector, DDP is combined. Cell proliferation was determined using MTT method. Changes of cell cycle and apoptotic rates were measured by flow cytometry. Western blot detected the effects of combining RKIP-pIRES2-EGFP with DDP upon MAPK pathway.
Result: (1) The recombinant plasmids were constructed successfully and confirmed by restiction enzyme digestion and sequencing results. After transfection with recombinant plasmid, green fluorescence were observed in Hep-2 cells under fluorescence microscope. And efficiency deteced by FCM was 81.53% at 24 hour post-transfection. Quantitative reverse transcriptase-polymerase chain and Western blotting were used to detect RKIP mRNA and protein expression. Treatment of Hep-2 with recombination plasmid resulted in a significant decrease or increase of RKIP expression at mRNA and protein level, which was more prominent in RKIP-shRNA501 group. (2) MTT assay demonstrated that cell growth was inhibited or promoted in transfected cells at 24 hour post-transfection. Cell growth decreased in high efficiency expression group, it is opposite in short hairpin RNA group, and 24 h, 48 h or 72 h growth inhibition or promotion rates of recombinant plasmid-transfection group were significantly different from that of the control group. FCM results showed that Hep-2 cells of high efficiency expression vector group increased in G1 stage, but Hep-2 cells of short hairpin RNA expression vector group decreased in G1 stage. Cell cycle was different between recombinant plasmid group and control group. Analysis of cell apoptosis showed obvious apoptotic peak in the cells transfected with high efficiency expression vector, but no obvious apoptotic peak was found in short hairpin RNA group. Matrigel invasion assay showed that cell invasiveness decreased in high efficiency expression group, it is opposite in short hairpin RNA group. Cell invasiveness was different between recombinant plasmid group and control group. Western blot detect the effects of different RKIP levels upon p-ERK protein. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP such as proliferation, cell cycle, apoptotic, invasiveness. (3) After transfection of Hep-2 cells with recombinant plasmid, DDP is combined. MTT assay demonstrated that cell growth was remarkably inhibited in RKIP-pIRES2-EGFP+DDP group. Cell growth is lower than that in any other three groups. FCM results showed cell apoptotic rate was significantly increased in RKIP-pIRES2-EGFP+DDP group. Cell apoptosis in RKIP-pIRES2- EGFP+DDP group is higher than that in any other three groups. Western blot detect the effects of combining RKIP-pIRES2-EGFP with DDP upon p-ERK protein. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP-pIRES2-EGFP combined with DDP.
Conclusion:
(1) RNAi to silence RKIP gene could down-regulate the express-on of RKIP and promote effect of hyperplasy and invasion level in Hep-2 cell. Applying molecular cloning technique to express RKIP, the situation is opposite. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP such as proliferation, cell cycle, apoptotic, invasiveness.
(2) The high efficiency expression of RKIP could not completely inhibit the hyperplasy of tumor cells, and the apoptotic-inducing effect was limited. The high efficiency expression of RKIP could coordinate with therapeutical effect by DDP. It could enhance the inhibition effect of hyperplasy in Hep-2 cell and promoted the apoptosis of laryngeal carcinoma cells, which indicates the high efficiency expression technique targeting RKIP might become a new strategy of gene therapy in laryngocarcinoma. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP-pIRES2-EGFP combined with DDP.
Head and neck squamous cell carcimoma (HNSCC) is a common malignant tumor in human body, which exhibits rapid growth, strong invasiveness and metastasis and has a high rate of recurrence. Carcinogenesis is a complicate staging process. From the point of view in molecular biology, major pathogenesy is reinforcement of oncogene and metastasis gene activity or inactivation of anti-oncogene and metastasis suppressor gene in human body interacting with various extent and internal factors. Combined radiotherapy and chemotherapy with operation has become a chief method and tendency for this disease. But management of HNSCC is a puzzled question in tumor therapy right along for its low sensitivity and strong resistance to chemotherapy. Meanwhile, the side effect and injury to normal tissue caused by chemotherapy can not be ignored. Therefore, it is urgent to find chemo sensitizer to enhance tumor chemo-sensitization and decrease injury in normal tissues. Due to the advantages of gene therapy, which include uneasy to produce drug resistance and strong specificity, it offers hope in this regard. Gene therapy involves the introduction of gene DNA into cells to exert effects by vector, which will selectively kill cancer cells with no toxicity to the surrounding non-malignant cells. The key point of gene therapy is the choice of target. Through such a process we can study and choose effective, low toxic cure medicine and method for oncotherapy, it is a new direction of oncotherapy to combine orthodox chemotherapy with gene therapy.
Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine binding protein family, a ubiquitously expressed and evolutionarily conserved group of proteins. In recent years, it has been reported that RKIP influence intracellular signaling cascades, cell cycle regulation, the suppression of metastasis, neuro generative processes, the modulation of emotions, and reproduction, and RKIP has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors and enhance tumor chemo-sensitization, but little is known about the role and the regulating mechanisms of RKIP in laryngocarcinoma.
RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing, resulting in the corresponding gene deletion phenotype. It is a hot spot in functional genomics investigation which develops gradually to become a critical technique for defining gene function.
In this study, we transfected recombinant plasmid to Hep-2 cell line with liposome to investigate the therapeutic value and possible mechanism of laryngeal carcinoma by regulating RKIP combined with DDP. Therefore, a theoretical basis and new utilization may be provided on enhancing combined therapeutic efficacy and improving prognosis of head and neck malignant tumors.
Objective: (1) To construct the recombinant plasmid of RKIP short hairpin RNA and high efficiency expression and to verify their efficacy. (2) To observe the effect of RKIP expression levels on cell proliferation, cell cycle, apoptosis, invasiveness and migration in Hep-2 cells, and investigate generation pathogenesy. (3) To observe the possibility of reducing laryngocarcinoma chemotherapy resistance and enhancing laryngocarcinoma sensitivity to DDP after high efficiency expression RKIP combined with DDP demonstrated by the hyperplasia and apoptosis of laryngeal squamous carcinoma cell, and investigate generation pathogenesy.
Method: (1) RKIP short hairpin RNA and high efficiency expression vector were constructed by molecular cloning technique and identified by enzyme digestion and sequencing. After transfection of Hep-2 cells with recombinant plasmid, transfection efficiency was observed and deteced by fluorescence microscope and FCM. Quantitative reverse transcriptase-polymerase chain and Western blotting were used to detect RKIP mRNA and protein expression. (2) After transfection of Hep-2 cells with recombinant plasmid, cell proliferation was determined using MTT method. Changes of cell cycle and apoptotic rates were measured by flow cytometry. Cell invasiveness was evaluated by Matrigel invasion assay. Western blot detected the effects of different RKIP levels upon MAPK pathway. (3) After transfection of Hep-2 cells with high efficiency expression vector, DDP is combined. Cell proliferation was determined using MTT method. Changes of cell cycle and apoptotic rates were measured by flow cytometry. Western blot detected the effects of combining RKIP-pIRES2-EGFP with DDP upon MAPK pathway.
Result: (1) The recombinant plasmids were constructed successfully and confirmed by restiction enzyme digestion and sequencing results. After transfection with recombinant plasmid, green fluorescence were observed in Hep-2 cells under fluorescence microscope. And efficiency deteced by FCM was 81.53% at 24 hour post-transfection. Quantitative reverse transcriptase-polymerase chain and Western blotting were used to detect RKIP mRNA and protein expression. Treatment of Hep-2 with recombination plasmid resulted in a significant decrease or increase of RKIP expression at mRNA and protein level, which was more prominent in RKIP-shRNA501 group. (2) MTT assay demonstrated that cell growth was inhibited or promoted in transfected cells at 24 hour post-transfection. Cell growth decreased in high efficiency expression group, it is opposite in short hairpin RNA group, and 24 h, 48 h or 72 h growth inhibition or promotion rates of recombinant plasmid-transfection group were significantly different from that of the control group. FCM results showed that Hep-2 cells of high efficiency expression vector group increased in G1 stage, but Hep-2 cells of short hairpin RNA expression vector group decreased in G1 stage. Cell cycle was different between recombinant plasmid group and control group. Analysis of cell apoptosis showed obvious apoptotic peak in the cells transfected with high efficiency expression vector, but no obvious apoptotic peak was found in short hairpin RNA group. Matrigel invasion assay showed that cell invasiveness decreased in high efficiency expression group, it is opposite in short hairpin RNA group. Cell invasiveness was different between recombinant plasmid group and control group. Western blot detect the effects of different RKIP levels upon p-ERK protein. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP such as proliferation, cell cycle, apoptotic, invasiveness. (3) After transfection of Hep-2 cells with recombinant plasmid, DDP is combined. MTT assay demonstrated that cell growth was remarkably inhibited in RKIP-pIRES2-EGFP+DDP group. Cell growth is lower than that in any other three groups. FCM results showed cell apoptotic rate was significantly increased in RKIP-pIRES2-EGFP+DDP group. Cell apoptosis in RKIP-pIRES2- EGFP+DDP group is higher than that in any other three groups. Western blot detect the effects of combining RKIP-pIRES2-EGFP with DDP upon p-ERK protein. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP-pIRES2-EGFP combined with DDP.
Conclusion:
(1) RNAi to silence RKIP gene could down-regulate the express-on of RKIP and promote effect of hyperplasy and invasion level in Hep-2 cell. Applying molecular cloning technique to express RKIP, the situation is opposite. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP such as proliferation, cell cycle, apoptotic, invasiveness.
(2) The high efficiency expression of RKIP could not completely inhibit the hyperplasy of tumor cells, and the apoptotic-inducing effect was limited. The high efficiency expression of RKIP could coordinate with therapeutical effect by DDP. It could enhance the inhibition effect of hyperplasy in Hep-2 cell and promoted the apoptosis of laryngeal carcinoma cells, which indicates the high efficiency expression technique targeting RKIP might become a new strategy of gene therapy in laryngocarcinoma. The changes of MAPK pathway were the whole or partial cause of the Hep-2 cells changes induced RKIP-pIRES2-EGFP combined with DDP.
引文
[1] Bernier I, Jolles P. Purification and characterization of a basic 23 kDa cytosolic protein from bovine brain[J]. Biochim Biophys Acta, 1984, 790: 174-181.
[2] Perry AC, Hall L, Bell AE, et al. Sequence analysis of a mammalian phospholipid- binding protein from testis and epididymis and its distribution between spermatozoa and extracellular secretions[J]. Biochem J, 1994, 301: 235-242.
[3] Bollengier F, Mahler A. Localization of the novel neuropolypeptide h3 in subsets of tissues from different species[J]. J Neurochem, 1988, 50: 1210-1214.
[4] Grandy DK, Hanneman E, Bunzow J, et al. Purification, cloning, and tissue distribution of a 23-kDa rat protein isolated by morphine affinity chromatography[J]. Mol Endocrinol, 1990, 4: 1370-1376.
[5] Hori N, Chae KS, Murakawa K, et al. A human cDNA sequence homologue of bovine phosphatidylethanolamine-binding protein[J]. Gene, 1994, 140: 293-294.
[6] Seddiqi N, Bollengier F, Alliel PM, et al. Amino acid sequence of the Homo sapiens brain 21-23-KDa protein (neuropolypeptide h3), comparison with its counterparts from Rattus norvegicus and Bos taurus species, and expression of its mRNA in different tissues[J]. J Mol Evol, 1994, 39:655-660.
[7] Frayne J, Ingram C, Love S, et al. Localisation of phosphatidylethanolamine-binding protein in the brain and other tissues of the rat[J]. Cell Tissue Res. 1999, 298: 415-423.
[8] Moore C, Perry AC, Love S, et al. Sequence analysis and immunolocalisation of phosphatidylethanolamine binding protein (PBP) in human brain tissue[J]. Brain Res Mol Brain Res, 1996, 37: 74-78.
[9] Goumon Y, Angelone T, Schoentgen F, et al. The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine- binding protein, possess a new biological activity on cardiac physiology[J]. J Biol Chem, 2004, 279: 13054-13064.
[10] Tsugu Y, Ojika K, Matsukawa N, et al. High levels of hippocampal cholinergicneurostimulating peptide (HCNP) in the CSF of some patients with Alzheimer’s disease[J]. Eur J Neurol, 1998, 5: 561-569.
[11] Mitake S, Ojika K, Hirano A. Hirano bodies and Alzheimer’s disease[J]. Kaohsiung J Med Sci, 1997, 13: 10-18.
[12] Mitake S, Katada E, Otsuka Y, et al. Possible implication of hippocampal cholinergic neurostimulating peptide (HCNP)-related components in Hirano body formation[J]. Neuropathol Appl Neurobiol, 1996, 22: 440-445.
[13] Yuasa H, Ojika K, Mitake S, et al. Age-dependent changes in HCNP-related antigen expression in the human hippocampus[J]. Brain Res Dev Brain Res, 2001, 127: 1-7.
[14] Thongboonkerd V, Klein JB. Practical bioinformatics for proteomics[J]. Contrib Nephrol, 2004, 141: 79-92.
[15] Yeung K, Seitz T, Li S, et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP[J]. Nature, 1999, 401:173-177.
[16] Corbit KC, Trakul N, Eves EM, et al. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein[J]. J Biol Chem, 2003, 278: 13061-13068.
[17] Lorenz K, Lohse MJ, Quitterer U. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2[J]. Nature, 2003, 426: 574-579.
[18] Yeung KC, Rose DW, Dhillon AS, et al. Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation[J]. Mol Cell Biol, 2001, 21: 7207-7217.
[19] Fu Z, Smith P C, Zhang L, et al. Effects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis[J]. J Natl Cancer Inst, 2003, 95: 878-889.
[20] Fu Z, Kitagawa Y, Keller ET, et al. Metastasis suppressor gene Raf kinase inhibitor protein (RKIP) is a novel prognostic marker in prostate cancer[J]. Prostate, 2006, 66: 248-256.
[21] Hagan S, Al-Mulla F, Mallon E, et al. Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis[J]. Clin Cancer Res, 2005, 11: 7392-7397.
[22] Schuierer MM, Bataille F, Hagan S, et al. Reduction in raf kinase inhibitor protein expression is associated within creased Ras-extra cellular signal-regulated kinase signal in melanoma cell lines[J]. Cancer Res, 2004, 64: 5186-5192.
[23] Chatterjee D, Bai Y, Wang Z, et a1. RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis[J]. J Biol Chem, 2004, 279: 17515-17523.
[24] Jazirehi AR, Vega MI, Chatterjee D, et al. Inhibition of the Raf-MEK1/2-ERK1/2 signaling pathway, Bcl-xL down-regulation, and chemosensitization of non-Hodgkin's lymphoma B cells by Rituximab[J]. Cancer Res, 2004, 64: 7117-7126.
[25] Bernier I, Tresca JP, Jolles P. Ligand-binding studies with a 23kDa protein purified from bovine brain cytosol[J]. Biochim Biophys Acta, 1986, 871: 19-23.
[26] Trakul N, Rosner MR. Modulation of the MAP kinase signaling cascade by Raf kinase inhibitory protein[J]. Cell Res, 2005, 15: 19-23.
[27] Chautard H, Jacquet M, Schoentgen F, et al. Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiae[J]. Eukaryot Cell, 2004, 3: 459-470.
[28] Gems D, Ferguson CJ, Maizels RM, et al. An abundant trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins[J]. J Biol Chem, 1995, 270: 18517-18522.
[29] Pikielny CW, Hasan G, Rouyer F, et al. Members of a family of Drosophila putative odorant-binding proteins are expressed indifferent subsets of olfactory hairs[J]. Neuron, 1994, 12: 35-49.
[30] Keller ET, Fu Z, Brennan M. The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein[J]. J Cell Biochem, 2005, 94: 273-278.
[31] Hickox DM, Gibbs G, Morrison JR, et al. Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2[J]. Biol Reprod, 2002, 67: 917-927.
[32] Wang X, Li N, Liu B, et al. A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-actived protein kinase pathway activation and phosphatidylethanolamine externalization[J]. JBiol Chem, 2004, 279: 45855-45864.
[33] Serre L, Vallee B, Bureaud N, et al. Crystal structure of the phosphatidylethanolamine- binding protein from bovine brain: a novel structural class of phospholipid-binding proteins[J]. Structure, 1998, 6: 1255-1265.
[34] Vallée BS, Tauc P, Brochon JC, et al. Behaviour of bovine phosphatidylethanolamine- binding protein with model membranes. Evidence of affinity for negatively charged membranes[J]. Eur J Biochem, 2001, 268:5831-5841.
[35] Frayne J, McMillen A, Love S, et al. Expression of phosphatidylethanolamine-binding protein in the male reproductive tract: immunolocalisation and expression in prepubertal and adult rat testes and epididymides[J]. Mol Rep Dev, 1998, 49: 454-460.
[36] Hengst U, Albrecht H, Hess D, et al. The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors[J]. J Biol Chem, 2001, 276: 535-540.
[37] Keller ET, Fu Z, Brennan M, et al. The role of Raf Kinase inhibitor protein (RKIP) in health and disease[J]. Biochem Pharmacol, 2004, 68: 1049-1053.
[38] Baccarini M. Second Nature: biological function of the Raf-1“kinases”[J]. FEBS Lett, 2005, 579: 3271-3277.
[39] Peyssonnaux C, Eychene A. The Raf/MEK/ERK pathway: new concepts of activation [J]. Biol Cell, 2001, 93: 53-62.
[40] Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions[J]. Biochem J, 2000, 351: 289-305.
[41] Schulze A, Lehmann K, Jefferies HB, et al. Analysis of the transcriptional program induced by Raf in epithelial cells[J]. Genes Dev, 2001, 15: 981-994.
[42] Yeung K, Janosch P, McFerran B, et al. Mechanism of suppression of the Raf/MEK/ extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein[J]. Mol Cell Biol, 2000, 20: 3079-3085.
[43] Trakul N, Menard RE, Schade GR, et al. Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation[J]. J Biol Chem, 2005, 280: 24931-24940.
[44] Yip-Schneider MT, Miao W, Lin A, et al. Regulation of the Raf-1 kinase domain byphosphorylation and 14-3-3 association[J]. Biochem J, 2000, 351: 151-159.
[45] Kroslak T, Koch T, Kahl E, et al. Human phosphatidylethanolamine-binding protein facilitates heterotrimeric G protein-dependent signaling[J]. J Biol Chem, 2001, 276: 39772-39778.
[46] Eves EM, Shapiro P, Naik K, et al. Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint[J]. Mol Cell, 2006, 23: 561-574.
[47] Li X, Stark GR. NF-κB-dependent signaling pathways[J]. Exp Hematol, 2002, 30: 285-296.
[48] Garg A, Aggarwal BB. Nuclear transcription factor-kappaB as a target for cancer drug development[J]. Leukemia, 2002, 16: 1053-1068.
[49] Greten FR, Eckmann L, Greten TF, et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer[J]. Cell, 2004, 118: 285-296.
[50] Pikarsky E, Porat RM, Stein I, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer[J]. Nature, 2004, 431: 461-466.
[51] Hinz M, Krappmann D, Eichten A, et al. NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition[J]. Mol Cell Biol, 1999, 19: 2690-2698.
[52] Kaltschmidt B, Kaltschmidt C, Hehner SP, et al. Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators[J]. Oncogene, 1999, 18: 3213-3215.
[53] Shishodia S, Aggarwal BB. Nuclear factor-kappaB: a friend or a foe in cancer[J]. Biochem Pharmacol, 2004, 68: 1071-1080.
[54] Ghosh S, May MJ, Kopp EB. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses[J]. Annu Rev Immunol, 1998, 16: 225-260.
[55] Karin M, Cao Y, Greten FR, et al. NF-κB in cancer: from innocent bystander to major culprit[J]. Nat Rev, 2002, 2: 301-310.
[56] Yamazaki T, Nakano H, Tsuchida S, et a1. Differentiation induction of human keratinocytes by phosphatidylethanolamine-binding protein[J]. J Biol Chem, 2004, 279:32191-32195.
[57] Zhang L, Fu Z, Binkley C, et al. Raf kinase inhibitory protein inhibits beta-cell proliferation[J]. Surgery, 2004, 136: 708-715.
[58] Baritaki S, Katsman A, Chatterjee D, et al. Regulation of tumor cell sensitivity to TRAIL-induced apoptosis by the metastatic suppressor Raf kinase inhibitor protein via Yin Yang 1 inhibition and death receptor 5 up-regulation[J]. J Immunol, 2007, 179: 5441-5453.
[59] Woods Ignatoski KM, Grewal NK, Markwart SM, et al. Loss of Raf kinase inhibitory protein induces radioresistance in prostate cancer[J]. Int J Radiat Oncol Biol Phys, 2008, 72: 153-160.
[60] Park S, Yeung ML, Yeung KC, et a1. RKIP downregulates B-Raf kinase activity in melanoma cancer cells[J]. Oncogene, 2005, 24: 3535-3540.
[61] Schuierer MM, Bataille F, Weiss TS, et al. Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma[J]. Oncol Rep, 2006, 16: 451-456.
[62] Li HZ, Wang Y, Gao Y, et al. Effects of raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer[J]. Mol Cancer Res, 2008, 6: 917-928.
[63] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391: 806-811.
[64] Jorgensen R. Altered gene expression in plants due to trans interactions between homologous genes[J]. Trends Biotechnol, 1990, 8: 340-344.
[65] Jorgensen RA, Cluster PD, Napoli CA, et al. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and singlecopy vs. complex T-DNA sequences[J]. Plant Mol Biol, 1996, 31: 957-973.
[66] Timmons L, Court DL, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene, 2001, 263: 103-112.
[67] Zamore PD, Tuschl T, Sharp PA, et al. RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals[J]. Cell, 2000, 101:25-33.
[68] Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs[J]. Genes Dev, 2001, 15: 188-200.
[69] Hutvágner G, Zamore PD. RNAi: nature abhors a double-strand[J]. Curr Opin Genet Dev, 2002, 12: 225-232.
[70] McCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice[J]. Nature, 2002, 418: 38-39.
[71]康洁,刘福林. RNAi的抗病毒作用及其机制[J].现代免疫学, 2004, 24: 439-441.
[72] Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs[J]. Cell, 2001, 107: 297-307.
[73]朱汝森,刘新光,梁念慈. RNAi实现策略的进展[J].国外医学临床生物化学与检验学分册, 2005, 25: 214-215.
[74] Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway[J]. Cell, 2001, 107: 309-321.
[75] Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs[J]. J Cell Science, 2001, 114: 4557- 4565.
[76] Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells[J]. Nucleic Acids Res, 2003, 31: 700-707.
[77] Caplen NJ, Fleenor J, Fire A, et al. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference[J]. Gene, 2000, 252: 95-105.
[78] Davenport RJ. Gene silencing: A faster way to shut down genes[J]. Science, 2001, 292: 1469-1471.
[79] Sijen T, Plasterk RH. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi[J]. Nature, 2003, 426, 310-314.
[80] Liu Q, Rand TA, Kalidas S, et al. R2D2, a bridge between the initiation and effectorSteps of the Drosophila RNAi Pathway[J]. Science, 2003, 301: 1921-1925.
[81] Baulcombe D. RNA silencing[J]. Current Biology, 2002, 12: 82-84.
[82] Dunoyer P, Lecellier CH, Parizotto EA, et al. Probing the microRNA and small interfering RNA Pathways with virus-encoded suppressors of RNA Silencing[J]. Plant Cell, 2004, 16: 1235-1250.
[83] Li WX, Li H, Lu R, et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[J]. PNAS, 2004, 101: 1350-1355.
[84] Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference[J]. Nature, 2002, 418: 435-438.
[85] Pennisi E. Evolution of developmental diversity meeting. RNAi takes Evo-Devo world by storm[J]. Science, 2004, 304: 384.
[86] Myers JW, Jones JT, Meyer T, et al. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing[J]. Nat Biotechnol, 2003, 21: 324- 328.
[87] Riddihough G. Molecular Biology: A Guide to Silence[J]. Science, 2004, 304: 931.
[88]高丽芳,徐德启,李峰,等. RNAi沉默STAT3基因对人前列腺癌PC3细胞裸鼠移植瘤生长的抑制作用[J].中华泌尿外科杂志, 2007, 28: 277.
[89] Crans-Vargas HN, Landaw EM, Bhatia S, et al. Expression of cyclic adenosine monophosphate response-element binding protein in acute leukemia[J]. Blood, 2002, 99: 2617-2619.
[90] Wang Q, Li M, Wang Y, et al. RNA interference targeting CML66, a novel tumor antigen, inhibits proliferation, invasion and metastasis of HeLa cells[J]. Cancer Lett, 2008, 269: 127-138.
[91] Wang Y, Zhu H, Quan L, et al. Downregulation of survivin by RNAi inhibits the growm of esophageal carcinoma cells[J]. Cancer Biol Ther, 2005, 4: 974-978.
[92] Fu GF, Lin XH, Han QW, et al. RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo[J]. Cancer Biol Ther, 2005, 4: 822-829.
[93] Zheng JN, Pei DS, Mao LJ, et al. Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encodingmRNA[J]. Cancer Gene Ther, 2009, 16: 20-32.
[94]于冬梅,郝立君,郭小英,等.人细胞周期蛋白cyclinD1基因RNAi表达载体的构建与鉴定[J].中国肿瘤, 2007, 16: 1008.
[95]李允光,濮德敏,刘雪梅,等. RNAi沉默环氧合酶-2基因对宫颈癌Hela细胞细胞周期影响的实验研究[J].中华肿瘤防治杂志, 2007, 14: 1535.
[96] Tsuchiya T, Okaji Y, Tsuno NH, et al. Targeting Idl and Id3 inhibits peritoneal metastasis of gastric cancer[J]. Cancer Sci, 2005, 96: 784-790.
[97] Shin J, Kim J, Ryu B, et al. Caveolin-l is associated with VCAM-l-dependent adhension of gastric cancer cells to endothelial cells[J]. Cell Physiol Biochem, 2006, 17: 211-220.
[98] Borkhardt A. Blocking oncogenes in malignant cells by RNA interference-new hope for a highly specific cancer treatment[J]. Cancer Cell, 2002, 2: 167-168.
[99] Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)[J]. Oncogene, 2002, 21: 5716-5724.
[100] Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Cell, 2002, 2: 243-247.
[101] De Souza NP, Alves G, Fiedler W. Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells[J]. Oncol Rep, 2006, 16: 423-428.
[102] Wu H, Hait WN, Yang JM, et al. Small interfering RNA-induced suppression of MDR1(P-Glycoprotein) restores sensitivity to multidrug-resistant cancer cells[J]. Cancer Res, 2003, 63: 1515-1519.
[103] Tuschl T, Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double- stranded RNA in vitro[J]. Genes Dev, 1999, 13: 3191-3197.
[104] Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture[J]. Nature, 2001, 411: 494-498.
[105] Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J]. EMBO J, 2001, 20: 6877-6888.
[106] Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structuralvariation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing[J]. Antisense Nucleic Acid Drug Dev, 2003, 13: 83-105.
[107] Chang F, Steelman LS, McCubrey JA, et a1. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention[J]. Leukemia, 2003, 17: 1263-1293.
[108] Kurihara N, Kubota T, Furukawa T, et al. Chemosensitivity testing of primary tumor cells from gastric cancer patients with liver metastasis can identify effective antitumor drugs[J]. Anticancer Res, 1999, 19: 5155-5158.
[109] Facchini LM, Penn LZ. The molecular role of Myc in growth and transformation: recent discoveries lead to new insights[J]. Faseb J, 1998, 12: 633-651.
[110] Grad JM, Zeng XR, Boise LH. Regulation of Bcl-xL: a little bit of this and a little bit of STAT[J]. Curr Opin Oncol, 2000, 12: 543-549.
[111] Borst P, Evers R, Kool M, et al. A family of drug transporters: the multidrug resistance- associated proteins[J]. J Natl Cancer Inst, 92: 1295-1302.
[112] Saleem A, Ibrahim N, Patel M, et al. Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning[J]. Cancer Res, 57: 5100-5106.
[2] Perry AC, Hall L, Bell AE, et al. Sequence analysis of a mammalian phospholipid- binding protein from testis and epididymis and its distribution between spermatozoa and extracellular secretions[J]. Biochem J, 1994, 301: 235-242.
[3] Bollengier F, Mahler A. Localization of the novel neuropolypeptide h3 in subsets of tissues from different species[J]. J Neurochem, 1988, 50: 1210-1214.
[4] Grandy DK, Hanneman E, Bunzow J, et al. Purification, cloning, and tissue distribution of a 23-kDa rat protein isolated by morphine affinity chromatography[J]. Mol Endocrinol, 1990, 4: 1370-1376.
[5] Hori N, Chae KS, Murakawa K, et al. A human cDNA sequence homologue of bovine phosphatidylethanolamine-binding protein[J]. Gene, 1994, 140: 293-294.
[6] Seddiqi N, Bollengier F, Alliel PM, et al. Amino acid sequence of the Homo sapiens brain 21-23-KDa protein (neuropolypeptide h3), comparison with its counterparts from Rattus norvegicus and Bos taurus species, and expression of its mRNA in different tissues[J]. J Mol Evol, 1994, 39:655-660.
[7] Frayne J, Ingram C, Love S, et al. Localisation of phosphatidylethanolamine-binding protein in the brain and other tissues of the rat[J]. Cell Tissue Res. 1999, 298: 415-423.
[8] Moore C, Perry AC, Love S, et al. Sequence analysis and immunolocalisation of phosphatidylethanolamine binding protein (PBP) in human brain tissue[J]. Brain Res Mol Brain Res, 1996, 37: 74-78.
[9] Goumon Y, Angelone T, Schoentgen F, et al. The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine- binding protein, possess a new biological activity on cardiac physiology[J]. J Biol Chem, 2004, 279: 13054-13064.
[10] Tsugu Y, Ojika K, Matsukawa N, et al. High levels of hippocampal cholinergicneurostimulating peptide (HCNP) in the CSF of some patients with Alzheimer’s disease[J]. Eur J Neurol, 1998, 5: 561-569.
[11] Mitake S, Ojika K, Hirano A. Hirano bodies and Alzheimer’s disease[J]. Kaohsiung J Med Sci, 1997, 13: 10-18.
[12] Mitake S, Katada E, Otsuka Y, et al. Possible implication of hippocampal cholinergic neurostimulating peptide (HCNP)-related components in Hirano body formation[J]. Neuropathol Appl Neurobiol, 1996, 22: 440-445.
[13] Yuasa H, Ojika K, Mitake S, et al. Age-dependent changes in HCNP-related antigen expression in the human hippocampus[J]. Brain Res Dev Brain Res, 2001, 127: 1-7.
[14] Thongboonkerd V, Klein JB. Practical bioinformatics for proteomics[J]. Contrib Nephrol, 2004, 141: 79-92.
[15] Yeung K, Seitz T, Li S, et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP[J]. Nature, 1999, 401:173-177.
[16] Corbit KC, Trakul N, Eves EM, et al. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein[J]. J Biol Chem, 2003, 278: 13061-13068.
[17] Lorenz K, Lohse MJ, Quitterer U. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2[J]. Nature, 2003, 426: 574-579.
[18] Yeung KC, Rose DW, Dhillon AS, et al. Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation[J]. Mol Cell Biol, 2001, 21: 7207-7217.
[19] Fu Z, Smith P C, Zhang L, et al. Effects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis[J]. J Natl Cancer Inst, 2003, 95: 878-889.
[20] Fu Z, Kitagawa Y, Keller ET, et al. Metastasis suppressor gene Raf kinase inhibitor protein (RKIP) is a novel prognostic marker in prostate cancer[J]. Prostate, 2006, 66: 248-256.
[21] Hagan S, Al-Mulla F, Mallon E, et al. Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis[J]. Clin Cancer Res, 2005, 11: 7392-7397.
[22] Schuierer MM, Bataille F, Hagan S, et al. Reduction in raf kinase inhibitor protein expression is associated within creased Ras-extra cellular signal-regulated kinase signal in melanoma cell lines[J]. Cancer Res, 2004, 64: 5186-5192.
[23] Chatterjee D, Bai Y, Wang Z, et a1. RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis[J]. J Biol Chem, 2004, 279: 17515-17523.
[24] Jazirehi AR, Vega MI, Chatterjee D, et al. Inhibition of the Raf-MEK1/2-ERK1/2 signaling pathway, Bcl-xL down-regulation, and chemosensitization of non-Hodgkin's lymphoma B cells by Rituximab[J]. Cancer Res, 2004, 64: 7117-7126.
[25] Bernier I, Tresca JP, Jolles P. Ligand-binding studies with a 23kDa protein purified from bovine brain cytosol[J]. Biochim Biophys Acta, 1986, 871: 19-23.
[26] Trakul N, Rosner MR. Modulation of the MAP kinase signaling cascade by Raf kinase inhibitory protein[J]. Cell Res, 2005, 15: 19-23.
[27] Chautard H, Jacquet M, Schoentgen F, et al. Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiae[J]. Eukaryot Cell, 2004, 3: 459-470.
[28] Gems D, Ferguson CJ, Maizels RM, et al. An abundant trans-spliced mRNA from Toxocara canis infective larvae encodes a 26-kDa protein with homology to phosphatidylethanolamine-binding proteins[J]. J Biol Chem, 1995, 270: 18517-18522.
[29] Pikielny CW, Hasan G, Rouyer F, et al. Members of a family of Drosophila putative odorant-binding proteins are expressed indifferent subsets of olfactory hairs[J]. Neuron, 1994, 12: 35-49.
[30] Keller ET, Fu Z, Brennan M. The biology of a prostate cancer metastasis suppressor protein: Raf kinase inhibitor protein[J]. J Cell Biochem, 2005, 94: 273-278.
[31] Hickox DM, Gibbs G, Morrison JR, et al. Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2[J]. Biol Reprod, 2002, 67: 917-927.
[32] Wang X, Li N, Liu B, et al. A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-actived protein kinase pathway activation and phosphatidylethanolamine externalization[J]. JBiol Chem, 2004, 279: 45855-45864.
[33] Serre L, Vallee B, Bureaud N, et al. Crystal structure of the phosphatidylethanolamine- binding protein from bovine brain: a novel structural class of phospholipid-binding proteins[J]. Structure, 1998, 6: 1255-1265.
[34] Vallée BS, Tauc P, Brochon JC, et al. Behaviour of bovine phosphatidylethanolamine- binding protein with model membranes. Evidence of affinity for negatively charged membranes[J]. Eur J Biochem, 2001, 268:5831-5841.
[35] Frayne J, McMillen A, Love S, et al. Expression of phosphatidylethanolamine-binding protein in the male reproductive tract: immunolocalisation and expression in prepubertal and adult rat testes and epididymides[J]. Mol Rep Dev, 1998, 49: 454-460.
[36] Hengst U, Albrecht H, Hess D, et al. The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors[J]. J Biol Chem, 2001, 276: 535-540.
[37] Keller ET, Fu Z, Brennan M, et al. The role of Raf Kinase inhibitor protein (RKIP) in health and disease[J]. Biochem Pharmacol, 2004, 68: 1049-1053.
[38] Baccarini M. Second Nature: biological function of the Raf-1“kinases”[J]. FEBS Lett, 2005, 579: 3271-3277.
[39] Peyssonnaux C, Eychene A. The Raf/MEK/ERK pathway: new concepts of activation [J]. Biol Cell, 2001, 93: 53-62.
[40] Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions[J]. Biochem J, 2000, 351: 289-305.
[41] Schulze A, Lehmann K, Jefferies HB, et al. Analysis of the transcriptional program induced by Raf in epithelial cells[J]. Genes Dev, 2001, 15: 981-994.
[42] Yeung K, Janosch P, McFerran B, et al. Mechanism of suppression of the Raf/MEK/ extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein[J]. Mol Cell Biol, 2000, 20: 3079-3085.
[43] Trakul N, Menard RE, Schade GR, et al. Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation[J]. J Biol Chem, 2005, 280: 24931-24940.
[44] Yip-Schneider MT, Miao W, Lin A, et al. Regulation of the Raf-1 kinase domain byphosphorylation and 14-3-3 association[J]. Biochem J, 2000, 351: 151-159.
[45] Kroslak T, Koch T, Kahl E, et al. Human phosphatidylethanolamine-binding protein facilitates heterotrimeric G protein-dependent signaling[J]. J Biol Chem, 2001, 276: 39772-39778.
[46] Eves EM, Shapiro P, Naik K, et al. Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint[J]. Mol Cell, 2006, 23: 561-574.
[47] Li X, Stark GR. NF-κB-dependent signaling pathways[J]. Exp Hematol, 2002, 30: 285-296.
[48] Garg A, Aggarwal BB. Nuclear transcription factor-kappaB as a target for cancer drug development[J]. Leukemia, 2002, 16: 1053-1068.
[49] Greten FR, Eckmann L, Greten TF, et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer[J]. Cell, 2004, 118: 285-296.
[50] Pikarsky E, Porat RM, Stein I, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer[J]. Nature, 2004, 431: 461-466.
[51] Hinz M, Krappmann D, Eichten A, et al. NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition[J]. Mol Cell Biol, 1999, 19: 2690-2698.
[52] Kaltschmidt B, Kaltschmidt C, Hehner SP, et al. Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators[J]. Oncogene, 1999, 18: 3213-3215.
[53] Shishodia S, Aggarwal BB. Nuclear factor-kappaB: a friend or a foe in cancer[J]. Biochem Pharmacol, 2004, 68: 1071-1080.
[54] Ghosh S, May MJ, Kopp EB. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses[J]. Annu Rev Immunol, 1998, 16: 225-260.
[55] Karin M, Cao Y, Greten FR, et al. NF-κB in cancer: from innocent bystander to major culprit[J]. Nat Rev, 2002, 2: 301-310.
[56] Yamazaki T, Nakano H, Tsuchida S, et a1. Differentiation induction of human keratinocytes by phosphatidylethanolamine-binding protein[J]. J Biol Chem, 2004, 279:32191-32195.
[57] Zhang L, Fu Z, Binkley C, et al. Raf kinase inhibitory protein inhibits beta-cell proliferation[J]. Surgery, 2004, 136: 708-715.
[58] Baritaki S, Katsman A, Chatterjee D, et al. Regulation of tumor cell sensitivity to TRAIL-induced apoptosis by the metastatic suppressor Raf kinase inhibitor protein via Yin Yang 1 inhibition and death receptor 5 up-regulation[J]. J Immunol, 2007, 179: 5441-5453.
[59] Woods Ignatoski KM, Grewal NK, Markwart SM, et al. Loss of Raf kinase inhibitory protein induces radioresistance in prostate cancer[J]. Int J Radiat Oncol Biol Phys, 2008, 72: 153-160.
[60] Park S, Yeung ML, Yeung KC, et a1. RKIP downregulates B-Raf kinase activity in melanoma cancer cells[J]. Oncogene, 2005, 24: 3535-3540.
[61] Schuierer MM, Bataille F, Weiss TS, et al. Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma[J]. Oncol Rep, 2006, 16: 451-456.
[62] Li HZ, Wang Y, Gao Y, et al. Effects of raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer[J]. Mol Cancer Res, 2008, 6: 917-928.
[63] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391: 806-811.
[64] Jorgensen R. Altered gene expression in plants due to trans interactions between homologous genes[J]. Trends Biotechnol, 1990, 8: 340-344.
[65] Jorgensen RA, Cluster PD, Napoli CA, et al. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and singlecopy vs. complex T-DNA sequences[J]. Plant Mol Biol, 1996, 31: 957-973.
[66] Timmons L, Court DL, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans[J]. Gene, 2001, 263: 103-112.
[67] Zamore PD, Tuschl T, Sharp PA, et al. RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals[J]. Cell, 2000, 101:25-33.
[68] Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs[J]. Genes Dev, 2001, 15: 188-200.
[69] Hutvágner G, Zamore PD. RNAi: nature abhors a double-strand[J]. Curr Opin Genet Dev, 2002, 12: 225-232.
[70] McCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice[J]. Nature, 2002, 418: 38-39.
[71]康洁,刘福林. RNAi的抗病毒作用及其机制[J].现代免疫学, 2004, 24: 439-441.
[72] Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs[J]. Cell, 2001, 107: 297-307.
[73]朱汝森,刘新光,梁念慈. RNAi实现策略的进展[J].国外医学临床生物化学与检验学分册, 2005, 25: 214-215.
[74] Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway[J]. Cell, 2001, 107: 309-321.
[75] Harborth J, Elbashir SM, Bechert K, et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs[J]. J Cell Science, 2001, 114: 4557- 4565.
[76] Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells[J]. Nucleic Acids Res, 2003, 31: 700-707.
[77] Caplen NJ, Fleenor J, Fire A, et al. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference[J]. Gene, 2000, 252: 95-105.
[78] Davenport RJ. Gene silencing: A faster way to shut down genes[J]. Science, 2001, 292: 1469-1471.
[79] Sijen T, Plasterk RH. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi[J]. Nature, 2003, 426, 310-314.
[80] Liu Q, Rand TA, Kalidas S, et al. R2D2, a bridge between the initiation and effectorSteps of the Drosophila RNAi Pathway[J]. Science, 2003, 301: 1921-1925.
[81] Baulcombe D. RNA silencing[J]. Current Biology, 2002, 12: 82-84.
[82] Dunoyer P, Lecellier CH, Parizotto EA, et al. Probing the microRNA and small interfering RNA Pathways with virus-encoded suppressors of RNA Silencing[J]. Plant Cell, 2004, 16: 1235-1250.
[83] Li WX, Li H, Lu R, et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing[J]. PNAS, 2004, 101: 1350-1355.
[84] Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference[J]. Nature, 2002, 418: 435-438.
[85] Pennisi E. Evolution of developmental diversity meeting. RNAi takes Evo-Devo world by storm[J]. Science, 2004, 304: 384.
[86] Myers JW, Jones JT, Meyer T, et al. Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing[J]. Nat Biotechnol, 2003, 21: 324- 328.
[87] Riddihough G. Molecular Biology: A Guide to Silence[J]. Science, 2004, 304: 931.
[88]高丽芳,徐德启,李峰,等. RNAi沉默STAT3基因对人前列腺癌PC3细胞裸鼠移植瘤生长的抑制作用[J].中华泌尿外科杂志, 2007, 28: 277.
[89] Crans-Vargas HN, Landaw EM, Bhatia S, et al. Expression of cyclic adenosine monophosphate response-element binding protein in acute leukemia[J]. Blood, 2002, 99: 2617-2619.
[90] Wang Q, Li M, Wang Y, et al. RNA interference targeting CML66, a novel tumor antigen, inhibits proliferation, invasion and metastasis of HeLa cells[J]. Cancer Lett, 2008, 269: 127-138.
[91] Wang Y, Zhu H, Quan L, et al. Downregulation of survivin by RNAi inhibits the growm of esophageal carcinoma cells[J]. Cancer Biol Ther, 2005, 4: 974-978.
[92] Fu GF, Lin XH, Han QW, et al. RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo[J]. Cancer Biol Ther, 2005, 4: 822-829.
[93] Zheng JN, Pei DS, Mao LJ, et al. Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encodingmRNA[J]. Cancer Gene Ther, 2009, 16: 20-32.
[94]于冬梅,郝立君,郭小英,等.人细胞周期蛋白cyclinD1基因RNAi表达载体的构建与鉴定[J].中国肿瘤, 2007, 16: 1008.
[95]李允光,濮德敏,刘雪梅,等. RNAi沉默环氧合酶-2基因对宫颈癌Hela细胞细胞周期影响的实验研究[J].中华肿瘤防治杂志, 2007, 14: 1535.
[96] Tsuchiya T, Okaji Y, Tsuno NH, et al. Targeting Idl and Id3 inhibits peritoneal metastasis of gastric cancer[J]. Cancer Sci, 2005, 96: 784-790.
[97] Shin J, Kim J, Ryu B, et al. Caveolin-l is associated with VCAM-l-dependent adhension of gastric cancer cells to endothelial cells[J]. Cell Physiol Biochem, 2006, 17: 211-220.
[98] Borkhardt A. Blocking oncogenes in malignant cells by RNA interference-new hope for a highly specific cancer treatment[J]. Cancer Cell, 2002, 2: 167-168.
[99] Wilda M, Fuchs U, Wossmann W, et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)[J]. Oncogene, 2002, 21: 5716-5724.
[100] Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Cell, 2002, 2: 243-247.
[101] De Souza NP, Alves G, Fiedler W. Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells[J]. Oncol Rep, 2006, 16: 423-428.
[102] Wu H, Hait WN, Yang JM, et al. Small interfering RNA-induced suppression of MDR1(P-Glycoprotein) restores sensitivity to multidrug-resistant cancer cells[J]. Cancer Res, 2003, 63: 1515-1519.
[103] Tuschl T, Zamore PD, Lehmann R, et al. Targeted mRNA degradation by double- stranded RNA in vitro[J]. Genes Dev, 1999, 13: 3191-3197.
[104] Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture[J]. Nature, 2001, 411: 494-498.
[105] Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J]. EMBO J, 2001, 20: 6877-6888.
[106] Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structuralvariation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing[J]. Antisense Nucleic Acid Drug Dev, 2003, 13: 83-105.
[107] Chang F, Steelman LS, McCubrey JA, et a1. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention[J]. Leukemia, 2003, 17: 1263-1293.
[108] Kurihara N, Kubota T, Furukawa T, et al. Chemosensitivity testing of primary tumor cells from gastric cancer patients with liver metastasis can identify effective antitumor drugs[J]. Anticancer Res, 1999, 19: 5155-5158.
[109] Facchini LM, Penn LZ. The molecular role of Myc in growth and transformation: recent discoveries lead to new insights[J]. Faseb J, 1998, 12: 633-651.
[110] Grad JM, Zeng XR, Boise LH. Regulation of Bcl-xL: a little bit of this and a little bit of STAT[J]. Curr Opin Oncol, 2000, 12: 543-549.
[111] Borst P, Evers R, Kool M, et al. A family of drug transporters: the multidrug resistance- associated proteins[J]. J Natl Cancer Inst, 92: 1295-1302.
[112] Saleem A, Ibrahim N, Patel M, et al. Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning[J]. Cancer Res, 57: 5100-5106.