摘要
第一部分LRIG3基因特异性RNA干扰真核表达载体的构建及胶质瘤细胞系稳定株筛选
目的构建LRIG3(leucine-rich repeats and immunoglobulin-like domains 3，LRIG3)基因特异的RNA干扰质粒，为探讨抑制LRIG3基因表达对脑胶质瘤细胞生物学行为调控的研究奠定基础。
方法根据GenBank数据库提供的LRIG3基因核苷酸序列，选择设计2条能转录短发卡RNA(Small hairpin RNA，shRNA)的DNA序列，命名为LRIG3-shRNA1、LRIG3-shRNA2，同时设计1条非特异性序列作为阴性对照，命名negative-shRNA。并与pGenesil2质粒载体连接，转化感受态大肠杆菌，挑选阳性克隆，抽取重组质粒，使用限制性内切酶SalⅠ酶切电泳，DNA测序鉴定。3种重组表达载体转染胶质瘤细胞系GL15细胞，用G418筛选后挑单克隆并扩增获得稳定株。RT-PCR和Western blot分别在mRNA和蛋白水平上检测LRIG3的表达。
结果重组质粒成功转化感受态大肠杆菌，经SalⅠ酶切琼脂糖凝胶电泳分析，表明寡核苷酸成功插入到预计位点，经测序鉴定，序列完全正确。G418筛选出稳定转染三种质粒的GL15细胞，转染pGenesil2-LRIG3-shRNA组细胞LRIG3 mRNA和蛋白表达明显低于转染pGenesil2-negative-shRNA组。
结论成功构建针对LRIG3基因的特异性shRNA真核表达载体，转染细胞后可抑制LRIG3基因表达，为进一步研究其基因功能奠定了基础。
第二部分LRIG3基因沉默对神经胶质瘤细胞GL15细胞系增殖及细胞周期和细胞凋亡的影响及其机制
目的探讨RNA干扰沉默LRIG3基因表达对神经胶质瘤细胞系GL15增殖及增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)和Ki-67表达，以及对细胞周期和细胞凋亡的影响及其作用机制。
方法用携带U6启动子和LRIG3特异性短发夹RNA(short hairpin RNA，shRNA)序列的质粒载体pGenesil2-LRIG3-shRNA1、pGenesil2-LRIG3-shRNA2及含非特异性shRNA编码序列的对照质粒pGenesil2-negative shRNA(neg)转染胶质瘤细胞系GL15，G418筛选出稳定株，逆转录聚合酶链反应(RT-PCR)和Western blot法检测LRIG3表达的改变，应用免疫组化SABC法检测对照组和实验组GL15细胞中PCNA和Ki-67的表达；应用流式细胞仪检测各组细胞的细胞周期变化，Annexin V-FITC／PI双标记分析细胞凋亡。
结果shRNA1及shRNA2组细胞LRIG3 mRNA水平与对照组相比分别下降了52．4％和63．8％，LRIG3蛋白水平分别下降了50．9％和67．4％。对照组细胞PCNA阳性率为(35．40±5．69)％，shRNA1及shRNA2组细胞PCNA阳性率分别为(72．13±5．64)％和(81．93±5．23)％，差异均有统计学意义(P＜0．01)。Ki-67阳性率在对照组细胞为(49．73±5．73)％，shRNA1及shRNA2组细胞分别为(82．27±5．50)％和(88．67±3．52)％，差异均有统计学意义(P＜0．01)。Ki-67表达与PCNA表达呈正相关(r=0．932，P＜0．001)。流式细胞仪分析显示，实验组的G2／M期细胞细胞百分率比对照组明显增加，细胞增殖指数显著增加(p＜0．01)；LRIG3基因表达下调后还具有显著的抗凋亡作用(p＜0．05)。
结论LRIG3特异性siRNA可明显抑制LRIG3基因的转录和表达，从而促进GL15胶质瘤细胞的增殖和使细胞周期阻滞在G2／M期并能抑制其凋亡。
第三部分下调LRIG3基因表达对EGFR信号通路和GL15细胞黏附、侵袭能力的影响及机制研究
目的研究RNA干扰LRIG3基因表达后对人胶质母细胞瘤GL15细胞系细胞黏附、侵袭能力的影响及其与表皮生长因子受体(EGFR)信号通路的关系。
方法用携带U6启动子和LRIG3特异性短发夹RNA(shRNA)序列的质粒载体pGenesil2-LRIG3-shRNA(siRNA)及含非特异性shRNA编码序列的对照质粒pGenesil2-negative shRNA(neg)转染GL15细胞系，G418(600ng／ml)筛选出稳定株，逆转录聚合酶链反应(RT-PCR)和Western-blot法检测LRIG3，EGFR和P-EGFR表达的改变。采用Transwell法和细胞基质黏附实验明确3株细胞在RNA干扰LRIG3基因表达后的侵袭能力及黏附能力，应用噻唑兰(MTT)法检测稳定转染后对GL15细胞增殖的影响。
结果两实验组细胞LRIG3 mRNA和蛋白水平与对照组相比分别下降了52．4％、63．8％和50．9％、67．4％，EGFR蛋白水平上升了40．4％和95．6％，P-EGFR蛋白水平上升了26．3％和81．2％。与对照组相比，两实验组GL15细胞的侵袭能力均显著提高，其细胞黏附性也显著增加，MTT结果显示实验组细胞增殖率高于对照组。
结论GL15细胞经RNA干扰基因表达后，LRIG3 mRNA和蛋白水平明显降低，同时EGFR和P-EGFR蛋白水平升高，从而提高细胞的侵袭能力和黏附性，同时可促进胶质瘤细胞的增殖。LRIG3可通过EGFR信号系统影响胶质瘤细胞的生长，有望成为胶质瘤分子治疗的靶点。
PartⅠConstruction of LRIG3 specific short-hairpin RNA expressingvector and screening of stably transfected cell clones
Objective To construct eukaryotic expression vectors of RNA interference specific forLRIG3 gene,and to screen the stably transfected cell clones.
Methods Genomic sequences of LRIG3 gene was retrieved from Genbank and cDNAwas designed encoding shRNA (small hairpin RNAs) for LRIG3.The cDNA wassynthesized and inserted into plasmid pGenesil2.Recombinant vectors were thentransformed into competent E.coli.The positive clones were selected and recombinantplasmids were extracted.The plasmids were digested with SalⅠand loaded in agarose gelelectrophoresis.The three shRNA vectors were transfected into GL15 by Metafectene.Thestably transfected cell clones were obtained after being screened with G418.Reversetranscriptase-polymerase chain reaction (RT-PCR) and Western blotting were performed toexamine the inhibitory effect at the RNA level and protein level.
Results The stably transfected pGenesil2-LRIG3-shRNA cell clones was significantlydown-regulated by siRNA as validated by RT-PCR and Western blotting.
Conclusions RNA interfering (RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG3 in glioma cell lines GL15.The stabletransfected cell clones was obtained for further study.
PartⅡEffects of RNAi-mediated gene silencing of LRIG3 expression onproliferation,cell cycle and cell apoptosis of GL15 cell lines and itsmechanisms
Objective To study the expression of proliferating cell nuclear antigen (PCNA),Ki-67 nuclear antigen,cell cycle and cell apoptosis in RNA interference (RNAi) - mediatedLRIG3 gene silencing Glioma cell and to investigate the correlation with tumor cellproliferation and its possible mechanisms.
Methods The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2were transfected into GL15 glioma cells respectively by Metafectine,and thetransfected-cells that stably suppressed LRIG3 expression were selected by G418.Thecontrol cells were transfected with negative shRNA (neg).The changes in LRIG3 mRNAand protein levels were measured by RT-PCR and Western blot.The expressions of PCNAand Ki-67 in GL 15 glioma cells were examined by SABC immunohistochemistry method.The apoptosis rate and cell cycle were analyzed by flow cytometry.
Results As compared with the negative shRNA-transfected GL 15 cells,LRIG3 mRNAexpression in GL15 cells transfected with pGenesil2-LRIG3-shRNA1 andpGenesil2-LRIG3-shRNA2 was silenced by 52.4%,63.8%,and LRIG3 protein expressionwas reduced by 50.9% and 67.4% respectively.The average PCNA positive staining cellwas (72.13±5.64) % in LRIG31-siRNA targeted cell and ( 81.93±5.23) % inLRIG32-siRNA targeted cell compared to negative control LRIG3 expressing cell (35.40±5.69) %(P＜0.01).Ki-67 labeling index (Ki-67 L I) from (82.27±5.50) % inLRIG31 -siRNA targeted cell to( 88.67±3.52) % in LRIG32-siRNA targeted cell and (49.73±5.73 ) % in negative controls ( P＜0.01).There was a good correlation between thePCNA L I and Ki-67 L I.Cell cycle analysis showed that silencing LRIG3 increased thepercentage of G2/M phase cells and the proliferation index significantly (P＜0.01).Silencing LRIG3 could inhibit the apoptosis of GL15 cells (P＜0.05).
Conclusions These findings suggest that the siRNA targeting LRIG3 gene shows adramatic inhibitory effect on RNA transcription and protein expression,then promoting theproliferation of GL15 cells,arresting GL15 cells in G2/M phase,and suppressing apoptosisof GL 15 cells.
PartⅢEffects of Down-regulating LRIG3 gene expression on theadhesion,invasion,and EGFR signaling pathways of GL15 cell lines andits mechanisms
Objective To explore the effects of RNA interference-mediated LRIG3 genesilencing on the adhesion and invasion of glioblastoma cell lines GL15 and to investigatethe correlation with EGFR signaling pathways.
Methods The plasmid pGenesil2-LRIG3-shRNA (siRNA) was transfected intoGL15 glioma cells by Metafectine,and the cells (siRNA) that stably suppress LRIG3expression were selected by G418.The control cells were transfected with negative shRNA(neg).The changes in LRIG3,EGFR and P-EGFR expression levels were analyzed byRT-PCR and Western blot.The changes of the GL15 cells adhesive and invasive abilitywere measured by cell adhesion assay and Transwell chamber.Cell proliferation wasdetected by MTT assay.
Results Compared with LRIG3 mRNA expression in the negative shRNA-treatedGL15 cells,the transcription after treatment with LRIG3-specific shRNA was silenced by52.4%、63.8%,and the expression of LRIG3 protein was reduced to 49.1%、32.6%.TheEGFR and P-EGFR protein level in pGenesil2-LRIG3-shRNA (siRNA) transfected cellswere significantly higher than that in negative shRNA (neg) transfected cells.Treatment ofGL15 cells with pGenesil2-LRIG3-shRNA can enhance adhesive and invasive ability.MTT assay showed that Cell proliferation was enhanced by LRIG3 shRNA transfection.
Conclusions Silencing LRIG3 expression could result in up-regulating of EGFR,then affect the downstream pathways of EGFR signaling,accordingly enhance the adhesiveand the invasive ability of GL15 cell,and can improve the proliferation of glioma GL15cells.LRIG3 could be a targeted protein for gene therapy of glioma.
引文
1. Andersson U., Gou D., Malmer B., et al. Epidermal growth factor receptor family(EGFR, ErbB2-4) in glioma and meningioma [J]. Acta Neuropathol(Berl), 2004, 108(2): 135-142.
2. Nilsson J, Vallbo C, Guo D, et al. Cloning, characterization, and expression of human LIG1 [J]. Biochem Biophys Res Commun, 2001, 284 (5): 1155-1161.
3. Ghiglione C, Carraway KL. Amundadottir LT., et al. The transmembrane molecule kekkonl acts in a feedback loop to negatively regulate the activity of the Drosophilia EGF receptor during Oogenesis [J]. Cell, 1999,96 (3): 847-856.
4. Suzuki Y., Miura H., Tanemura A., et al. Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia [J]. FEBS Lett, 2002, 521(5): 67-71.
5. Holmlund C, Nilsson J, Guo D, et al. Characterization and tissue-specific expression of human LRIG2 [J]. Gene, 2004, 332 (4): 35-43.
6. Guo D, Holmlund C, Henriksson R, et al. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues, and a homolog in Ascidiacea [J]. Genomics, 2004, 84 (3): 157-165.
7. Suzuki Y, Sato N, Tohyama M, et al. cDNA cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains.J Biol Chem, 1996, 271:22522-22527.
8. Gur G, Rubin C, Katz M, et al. LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation. EMBO J, 2004,23: 3270-3281.
9. Laederich MB, Funes DM, Yen L, et al. The leucinerich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. J Biol Chem, 2004, 279: 47050-47056.
10. Goldoni S, Iozzo RA, Kay P, et al. A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity. Oncogene, 2007, 26:368-381.
11. Hakan H, Roger H. LRIG inhibitors of growth factor signaling- double-edged swords in human cancer?. Eur J Cancer,2007, 43:676-682.
12. Yarden Y. The EGFR family and its ligands in human cancer: signaling mechanisms and therapeutic opportunities. European Journal of Cancer, 2001, 37:3-8.
13. Kondo I, Shimizu N. Mapping of the human gene for epidermal growth factor receptor (EGFR) on the pl3 leads to q22 region of chromosome 7. Cytogenet Cell Genet, 1983, 35:9.
14. Elbashir SM, Harborth J, LendeckelW, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411(6836): 494-498.
15. Harmon GJ, Conklin D S. RNA interference by short hairpin RNAs expressed in vertebrate cells [J]. Methods Mol Biol, 2004, 257 (1): 255-266.
16. Brummelkamp T R, Bernards R. A systemfor stable expression of short interfering RNAs in mammalia cells [J]. Science, 2002,296 (4): 550-553.
17. MiyagishiM, Taira K. U6 promoter2driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat Biotechnol, 2002,19 (5): 497-500.
1. Suzuki Y, Sato N, Tohyama M, et al cDNA cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains [J]. Biol Chem, 1996,271: 22522-22527.
2. Nilsson J, Vallbo C, Guo D, et al Cloning, characterization, and expression of human LIG1 [J]. Biochem Biophys Res Commun, 2001,284 : 1155-1161.
3. Holmlund C, Nilsson J, Guo D, et al Characterization and tissue-specific expression of human LRIG2 [J]. Gene, 2004, 332 : 35-43.
4. Guo D, Holmlund C, Henriksson R, et al The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues, and a homolog in Ascidiacea [J]. Genomics, 2004, 84 : 157-165.
5. Gur G, Rubin C, Katz M, et al LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation [J]. EMBO J, 2004, 23 : 3270-3281.
6. Laederich MB, Funes-Duran M, Yen L, et al The leucinerich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases [J]. J Biol Chem, 2004, 279:47050-47056.
7. Goldoni S, Iozzo RA, Kay P, et al A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity [J]. Oncogene, 2006, doi: 10.1038/ sj.one.1209803.
8. Hedman H, Nilsson J, Guo D, et al Is LRIG1 a tumour suppressor gene at chromosome 3pl4.3? [J]. Acta Oncol, 2002,41: 352-354.
9. Thomasson M, Hedman H, Guo D, et al LRIG1 and epidermal growth factor receptor in renal cell carcinoma: a quantitative RT-PCR and immunohistochemical analysis [J]. Br J Cancer, 2003, 89 : 1285-1289.
10. Tanemura A, Nagasawa T, Inui S, et al LRIG-1 provides a novel prognostic predictor in squamous cell carcinoma of the skin: immunohistochemical analysis for 38 cases [J ]. Dermatol Surg, 2005, 31:423-430.
11.Hakan Hedman, Roger Henriksson LRIG inhibitors of growth factor signaling-double-edged swords in human cancer?[J ]. Eur J Cancer, 43(2007),676-682.
12.蔡明俊,雷霆,郭东生RNA干扰技术在胶质瘤治疗中的应用[J].中国临床神经外科杂志,2007, 12 (5):319-321.
13.Yuan B, Latek R, Hossbach M, et al siRNA Selection Server: an automated siRNA oligonucleotide prediction server[J].Nucleic Acids Res, 2004, Vol, 32:130-134.14.Tuchl T Expanding small RNA interference[J ]. Nature Biotechnology,2002, 20(5):446-448.
15.Andersson U., Gou D., Malmer B., et al Epidermal growth factor receptor family(EGFR, ErbB2-4) in glioma and meningioma[J].Acta Neuropathol(Berl), 2004,108(2):135-142.
16.Ghiglione C., Carraway KL., Amundadottir LT., et al The transmembrane molecule kekkonl acts in a feedback loop to negatively regulate the activity of the Drosophilia EGF receptor during Oogenesis[J].Cell, 1999, 96:847.
17.Suzuki Y., Miura H., Tanemura A., et al Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia[J ]. FEBS Lett, 521(2002):67-71.
18.叶飞,郭东生,牛洪泉等.LRIG1 cDNA诱导人胶质瘤细胞系H4凋亡的分子机制[J].癌症,2004, 23(10):1149-1154.
19.Elbashir SM, Harborth J, Lendeckel W, et al Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature, 2001,411(6836):494-498.
20.Hannon GJ,Conklin D S RNA interference by short hairpin RNAs expressed in vertebrate cells [J].Methods Mol Biol, 2004, 257 (1):255.
21.Brummelkamp T R,Bernards R A systemfor stable expression of short interfering RNAs in mammalia cells [J 1. Science, 2002, 296:550.
22. MiyagishiM, Taira K U6 promoter2driven siRNAs with four uridine 2'overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat Biotechnol, 2002,20:4972500.
1.叶飞,郭东升,牛洪泉,等.LRIG1 cDNA诱导人胶质瘤细胞系H4凋亡的分子机制[J].癌症,2004,23(10):1149-1154.
2.Nilsson J, Vallbo C, Guo D, et al. Cloning, characterization, and expression of human LIG1 [J]. Biochem Biophys Res Commun, 2001,284(5):1155-1161.
3.Holmlund C, Nilsson J, Guo D, et al. Characterization and tissue-specific expression of human LRIG2 [J]. Gene, 2004,332(5):35-43.
4.Guo D, Holmlund C, Henriksson R, et al. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues, and a homolog in Ascidiacea[J]. Genomics, 2004,84(1):157-165.
5.Takahashi N, Takahashi Y, Putnam FW. Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum. Proc Natl Acad Sci U S A 1985,82:1906-10.
6.Kobe B, Deisenhofer J. Proteins with leucine-rich repeats. Curr Opin Struct Biol 1995,5 :409-416.
7.Winans KA, Hashimoto C. Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein. Mol Biol Cell 1995,6:587-596.
8.Malim MH, Bohnlein S, Hauber J, Cullen BR. Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function. Cell1989,58 ,205-14.
9.Miyachi K, Fritzler MJ, Tan EM. Autoantibody to a nuclear antigen in proliferating cells [J]. J Immunol, 1978,121(6):2228-2234.
10.Bravo R, Frank R, Blundell PA, et al. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta [J]. Nature, 1987,326(4):515-517.
11.Louis DN, Edgerton S, Thor AD, et al. Proliferating cell nuclear antigen and Ki-67 immunohistochemistry in brain tumors: a comparative study [J]. Acta Neuropathol,1991,81(6):675-679.
12. Yao T, Tsuneyoshi M, Matsumoto T, et al. Depressed alenoma of the colorectum:analysis of proliferative activity using Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) [J]. Pathol Int, 1994,44 (7):520-527.
13. Gerdes J, Schwab U, Lemke H, et al. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation [J]. Int J Cancer, 1983,31(1):13-20.
14. Scholzen T, Gerdes. The Ki-67 protein: from the known and the unknown [J]. J Cell Physiol, 2000,182(3):311-322.
15. Neder L, Colli BO, Machado HR, et al. MIB-1 labeling index in astrocytic tumors-a clinicopathologic study [J]. Clin Neuropathol, 2004,23(6):262-270
16. Zuber P, Hamou MF, Tribolet N. Identification of proliferating cells in human gliomas using the monoclonal antibody Ki-67 [J]. Neurosurgery, 1988,22(2):364-368.
17. Esteban F, de Vega DS, Garcia R, et al. DNA content by flow cytometry in gastric carcinoma : pathology, ploidy and prognosis. Hepatogastroenterology. 1999, 46: 2041.
[1] Guo D, Holmlund C, Henriksson R, et al. The LRIG gene family has three vertebrate paralogs widely expressed in human and mouse tissues, and a homolog in Ascidiacea. Genomics, 2004, 84:157-165.
[2] Suzuki Y, Sato N, Tohyama M, et al. cDNA cloning of a novel membrane glycoprotein that is expressed specifically in glial cells in the mouse brain. LIG-1, a protein with leucine-rich repeats and immunoglobulin-like domains. J Biol Chem, 1996, 271:22522-22527.
[3] Nilsson J, Vallbo C, Guo D, et al. Cloning, characterization, and expression of human LIG1. Biochem Biophys Res Commun, 2001,284:1155-1161.
[4] Holmlund C, Nilsson J, Guo D, et al. Characterization and tissue-specific expression of human LRIG2. Gene, 2004, 332:35-43.
[5] Gur G, Rubin C, Katz M, et al. LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation. EMBO J, 2004, 23: 3270-3281.
[6] Laederich MB, Funes DM, Yen L, et al. The leucinerich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. J Biol Chem, 2004, 279: 47050-47056.
[7] Goldoni S, Iozzo RA, Kay P, et al. A soluble ectodomain of LRIG 1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity. Oncogene, 2007,26:368-381.
[8] Hakan H, Roger H. LRIG inhibitors of growth factor signaling-double-edged swords in human cancer?. Eur J Cancer,2007,43:676-682.
[9] Yarden Y. The EGFR family and its ligands in human cancer: signaling mechanisms and therapeutic opportunities. European Journal of Cancer, 2001,37:3-8.
[10] Kondo I, Shimizu N. Mapping of the human gene for epidermal growth factor receptor (EGFR) on the p13 leads to q22 region of chromosome 7. Cytogenet Cell Genet, 1983,35:9.
[11]蔡明俊,雷霆,郭东生.RNA干扰技术在胶质瘤治疗中的应用.中国临床神经外科杂志,2007,12: 319-321.
[12]吕佳音,高忠礼,王金成,等.RNA干扰沉默mdm2治疗骨肉瘤的实验研究.中华肿瘤杂志,2008, 30: 502-505.
1 Napoli C, Lemieux C, Jorgensen R. Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans [J]. Plant Cell, 1990,2:279-289.
2 Guo S, Kempheus KJ. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed[J]. Cell, 1995,81 1611-620.
3 Andrew Fire, SiQun Xu, Mary K.Montgomery, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391 (6669) -806-811.
4 Carl D. Novina and Phillip A. Sharp. The RNAi revolution[J]. Nature, 2004, 430 (6995): 161-164.
5 Brummelkamp TR, Bernards R, Agami R. A system for stable expression short interfering RNAs in mammalian cells[J]. Science, 2002,296 (5567): 550-553.
6 Hannon GJ. RNA interference [J]. Nature, 2002,418(6894): 244-251.
7 Caplen NJ, Fleenor J, Fire A, et al. DsRNA-mediated gene silencing in cultured Drosophila cells[J].Gene, 2000,252: 95-105.
8 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.
9 Nykanen A, Haley B, Zamore. PD, et al.ATP requirements and small interfering structure in the RNA interference pathway [J]. Cell, 2001, 107(3): 309-321.
10 M. Miyagishi, K. Taira. Development and application of siRNA expression vector[J]. Nucleic Acids Res. Suppl, (2002) 113-114.
11 M. Miyagishi, M. Hayashi, K. Taira. Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells[J]. Antisense Nucleic Acid Drug Dev, 13 (2003) 1-7.
12 M. Hamada, T. Ohtsuka, R. Kawaida, et al. Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs[J]. Antisense Nucleic Acid Drug Dev, 12(2002)301-309.
13 Brummelkamp TR, Bernards R,Aagmi R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Press, 2002,2 (3): 243- 247.
14 Dong Hokim, Mark A behlke, Scott D Rose, et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy[J]. Nat. Biotechnol, 2005, 23(2): 222-226.
15 Despina Siolas, Caca Lerner, Julija Burchard et al. Synthetic shRNAs as potent RNAi triggers [J]. Nat. Biotechnol, 2005, 23(2): 227-231.
16 Rahaman SO, Vogelbaum MA, Haque SJ. Aberrant Stat3 signaling by interleukin-4 in malignant glioma cells: involvement of IL-13Ralpha2[J]. Cancer Res, 2005, 65(7) :2956-2963.
17 Huang A, Ho CS, Ponzielli R, et al. Identification of a novel c-Myc protein interactor, JPO2, with transforming activity in medulloblastoma cells[J]. Cancer Res, 2005 ,65(13): 5607-5619.
18 James R Van Brocklyn, Catherine A Jackson, Dennis K Pearl, Mark S Kotur, et al. Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines[J]. J Neuropathol Exp Neurol, 2005, 64(8): 695-705.
19 Okay Saydam, Daniel L. Glauser, Irma Heid, et al. Herpes simplex virus 1 amplicon vector-mediated siRNA targeting epidermal growth factor receptor inhibits growth of human glioma cells in vivo[J]. Molecular Therapy, 2005,12(5): 803-812.
20 Purow BW, Haque RM, Noel MW, et al. Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation[J]. Cancer Res, 2005, 65(6): 2353-2363.
21 Zhang Y, Zhang YF, Bryant, et al. Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer[J]. Clin Cancer Res, 2004, 10(11): 3667-3677.
22 Jo Whelan. First clinical data on RNAi[J]. DDT, 2005,10(15): 1014-1015.