RNA干扰HBx稳定表达肝癌细胞株细胞增殖及化疗增敏作用的研究
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摘要
背景及目的:
目前大量实验研究证实HBx片断可在多数并发乙肝病毒感染的肝癌组织中检测到,它是肝细胞发生恶性转化的主要因素之一,并可提高肝癌细胞的增殖活性,促进肝癌细胞快速增长。因此,高效特异的基因治疗新技术RNA干扰靶向沉默HBx基因的应用是肝癌治疗的一种新方法。另一方面,化疗仍是目前肝癌治疗重要手段之一,但多种化疗药物耐药性是肝癌治疗失败的主要原因。而相关研究表明,HBx片断可通过特异MAPK途径上调多药耐药基因的表达,并使抗凋亡信号占主导地位,抑制化疗药物诱导的肝癌细胞凋亡。因此我们拟采用针对HBx的RNA干扰技术与常用的肝癌化疗药物或抗肿瘤药协同应用,探讨RNA干扰技术和传统的治疗方法联合应用对肝癌细胞的生长抑制作用的影响,并选择高效的治疗组合,研究其凋亡机制。
方法:
1.鉴定转染无关的对照序列(HK3细胞)和针对X基因shRNA(21543细胞)的稳定肝癌细胞株:复苏三种肝癌细胞(MHCC97-H,HK3,21543细胞),在含有一定浓度G418的高糖培养基中培养一段时间后,倒置荧光显微镜观察GFP的表达,初步估计转染效率并拍照。
2.RT-PCR检测RNA干扰对HBx基因mRNA沉寂的程度:收集细胞提取总RNA,加用两对不同引物参照说明进行RT-PCR,根据凝胶电泳中内源基因片段条带的强弱,来判定模板的量,进而判断HBX基因的mRNA被siRNA沉寂的程度。
3.观察RNA干扰后对细胞生长的影响:利用CCK8(Cell countingkit-8)测定光密度值,绘制三种不同细胞6天的不同增殖曲线。
4.流式细胞仪检测细胞周期的变化。
5.绘制加用三种不同浓度的5-氟脲嘧啶,顺铂,α-干扰素的肝癌细胞的生长曲线,选择最佳组合。
6.TUNEL细胞凋亡检测试剂盒检测细胞凋亡。
结果:
1.复苏后细胞形态和GFP表达强度差别不大。
2.RT-PCR结果显示相对于MHCC-97H细胞,21543细胞的HBxmRNA水平的下调显著,HK3细胞的HBx mRNA水平的下调不明显。
3.靶向HBX的RNA干扰后的肝癌细胞(21543细胞)较原肝癌细胞(MHCC97-H细胞)和转染无关对照序列的肝癌细胞(HK3细胞)增殖明显减慢,后两种细胞差别不显著。
4.21543细胞的细胞周期显示RNA干扰后的肝癌细胞延缓进入S期,增殖活性明显降低。
5.三种不同细胞加用三种不同浓度的5-氟脲嘧啶,顺铂后细胞生长明显减慢,并呈浓度依赖性,以RNA干扰HBx后的肝癌细胞(21543细胞)生长抑制最明显,加用干扰素后细胞抑制不明显。
6.相同浓度的化疗药物5-氟脲嘧啶作用上述三种不同肝癌细胞均可引起细胞凋亡,以RNA干扰后的肝癌细胞(21543细胞)最为明显。
结论:
1.RNA干扰HBx可明显抑制肝癌细胞的增长,细胞周期发生改变。
2.RNA干扰HBx后的肝癌细胞可明显增强化疗敏感性。
3.RNA干扰HBx和化疗药物二者联合应用肝癌细胞凋亡更为明显,细胞增殖明显减慢。
Background and Objective:
There are plenty of experiments which have proved that HBx can be detected in most of the Hepatocellular carcinoma (HCC) tissue infected by hepatitis B virus. HBx is one of the principal factors of hepatocyte malignant transformation,it can increase proliferation activity of Hepatocellular carcinoma cell and promote its growth quickly.RNA interference targeting to HBx is a new approach with high performance and specificity in the gene therapy of liver cancer .Chemotherapy is still one of important methods to treat the Hepatocellular carcinoma now.Neverthless, drug resistance in chemotherapeutics is a primary cause of treatment failure in liver cancer. Related study make clear that HBx can upregurate the expression of multidrug resistance gene through special MAPK pathway . Then anti-apoptosis signal ocuppy the manage position and supress the apoptosis of Hepatocellular carcinoma cell induced by the chemotheraputics.For this reason ,we plan to adopt the RNA interference to aim directly at HBx and traditionary antinelplastic agent of liver cancer.We shoud investigate the influence of association RNA interference with traditional therapy.We should select the therapy combination of high performance and study the apoptosis mechanism.
Methods:
1.Identity the stable Hepatocellular carcinoma cells transfected by shRNA aimming at HBx together with independent control series:we should reanimate three kinds of Hepatocellular carcinoma cell(MHCC97-H,HK3,21543),cultivate them in the high glucose DMEM with G418 for some days,then observe the expresion of GFP in cells through inversion fluorescent microscope and initial to estimate the transfection efficiency ,take photographs at last.
2.Detect the extent of HBx gene by RNA interference by semipuanitative RT-PCR : collect three kinds of cell and extract total RNA,add two different pairs of primer and carry out RT-PCR in accordance with illustration,On the basis of endogenous gene strap in the gel electrophoresis,we assessment the tempate quantity and judge the extent of HBx gene by RNA interference.
3.0bserve the influence of cell growth through RNA interference:we should utilize the Cell counting kit-8 and evaluate optical density value ,then we draw the growth curve of three kinds of cell in six days.
4.Detect the diversity of cell cycle by flow cytometry
5.Draw the growth curve of three kinds of cell after add different density of flurouracil ,cisplatin andα-interferon and select the best combination.
6.Detect the cell apoptosis by TUNEL apoptosis detection kit
Result:
1 .There are no difference in the shape and GFP expression intensity in two transfected cell after resuscitation.
2.The result of RT-PCR demonstrate that the HBX mRNA level of 21543 cell down regulate, the HBx mRNA level of HK3 cell is not different with MHCC97-H cell.
3.The growth of 21543 cell are slower than MHCC97-H cell and HK3 cell obviously. The latter two cells are not different notably.
4.The cell cycle of 21543 cell showed that Hepatocellular carcinoma cell through RNA interference targeting to HBx delay to goto S stage,then proliferation activity degrade obviously.
5.Three kinds of cell adding different density of flurouracil ,cisplatin grows slower than the original cell. The growth inhibiting is dependent on the density of drug and the growth inhibiting of 21543 cell is the most obvious.That of three kind of cell adding toα-interferon is not obvious.
6. Flurouracil induce apoptosis in all kinds of cell . The extent of apoptosis in 21543 cell is most obvious.
Conclusion: 1. RNA interference targeting to HBx can supress the growth of hepatocellular carcinoma cell.
2. Hepatocellular carcinoma cell through RNA interference targeting to HBx can intensify chemo-sensitivity.
3. Combination RNA interference targeting to HBx with chemotherapeutics can induced apoptosis in more hepatocellular carcinoma cell.Cell proliferation step down accordingly.
目前大量实验研究证实HBx片断可在多数并发乙肝病毒感染的肝癌组织中检测到,它是肝细胞发生恶性转化的主要因素之一,并可提高肝癌细胞的增殖活性,促进肝癌细胞快速增长。因此,高效特异的基因治疗新技术RNA干扰靶向沉默HBx基因的应用是肝癌治疗的一种新方法。另一方面,化疗仍是目前肝癌治疗重要手段之一,但多种化疗药物耐药性是肝癌治疗失败的主要原因。而相关研究表明,HBx片断可通过特异MAPK途径上调多药耐药基因的表达,并使抗凋亡信号占主导地位,抑制化疗药物诱导的肝癌细胞凋亡。因此我们拟采用针对HBx的RNA干扰技术与常用的肝癌化疗药物或抗肿瘤药协同应用,探讨RNA干扰技术和传统的治疗方法联合应用对肝癌细胞的生长抑制作用的影响,并选择高效的治疗组合,研究其凋亡机制。
方法:
1.鉴定转染无关的对照序列(HK3细胞)和针对X基因shRNA(21543细胞)的稳定肝癌细胞株:复苏三种肝癌细胞(MHCC97-H,HK3,21543细胞),在含有一定浓度G418的高糖培养基中培养一段时间后,倒置荧光显微镜观察GFP的表达,初步估计转染效率并拍照。
2.RT-PCR检测RNA干扰对HBx基因mRNA沉寂的程度:收集细胞提取总RNA,加用两对不同引物参照说明进行RT-PCR,根据凝胶电泳中内源基因片段条带的强弱,来判定模板的量,进而判断HBX基因的mRNA被siRNA沉寂的程度。
3.观察RNA干扰后对细胞生长的影响:利用CCK8(Cell countingkit-8)测定光密度值,绘制三种不同细胞6天的不同增殖曲线。
4.流式细胞仪检测细胞周期的变化。
5.绘制加用三种不同浓度的5-氟脲嘧啶,顺铂,α-干扰素的肝癌细胞的生长曲线,选择最佳组合。
6.TUNEL细胞凋亡检测试剂盒检测细胞凋亡。
结果:
1.复苏后细胞形态和GFP表达强度差别不大。
2.RT-PCR结果显示相对于MHCC-97H细胞,21543细胞的HBxmRNA水平的下调显著,HK3细胞的HBx mRNA水平的下调不明显。
3.靶向HBX的RNA干扰后的肝癌细胞(21543细胞)较原肝癌细胞(MHCC97-H细胞)和转染无关对照序列的肝癌细胞(HK3细胞)增殖明显减慢,后两种细胞差别不显著。
4.21543细胞的细胞周期显示RNA干扰后的肝癌细胞延缓进入S期,增殖活性明显降低。
5.三种不同细胞加用三种不同浓度的5-氟脲嘧啶,顺铂后细胞生长明显减慢,并呈浓度依赖性,以RNA干扰HBx后的肝癌细胞(21543细胞)生长抑制最明显,加用干扰素后细胞抑制不明显。
6.相同浓度的化疗药物5-氟脲嘧啶作用上述三种不同肝癌细胞均可引起细胞凋亡,以RNA干扰后的肝癌细胞(21543细胞)最为明显。
结论:
1.RNA干扰HBx可明显抑制肝癌细胞的增长,细胞周期发生改变。
2.RNA干扰HBx后的肝癌细胞可明显增强化疗敏感性。
3.RNA干扰HBx和化疗药物二者联合应用肝癌细胞凋亡更为明显,细胞增殖明显减慢。
Background and Objective:
There are plenty of experiments which have proved that HBx can be detected in most of the Hepatocellular carcinoma (HCC) tissue infected by hepatitis B virus. HBx is one of the principal factors of hepatocyte malignant transformation,it can increase proliferation activity of Hepatocellular carcinoma cell and promote its growth quickly.RNA interference targeting to HBx is a new approach with high performance and specificity in the gene therapy of liver cancer .Chemotherapy is still one of important methods to treat the Hepatocellular carcinoma now.Neverthless, drug resistance in chemotherapeutics is a primary cause of treatment failure in liver cancer. Related study make clear that HBx can upregurate the expression of multidrug resistance gene through special MAPK pathway . Then anti-apoptosis signal ocuppy the manage position and supress the apoptosis of Hepatocellular carcinoma cell induced by the chemotheraputics.For this reason ,we plan to adopt the RNA interference to aim directly at HBx and traditionary antinelplastic agent of liver cancer.We shoud investigate the influence of association RNA interference with traditional therapy.We should select the therapy combination of high performance and study the apoptosis mechanism.
Methods:
1.Identity the stable Hepatocellular carcinoma cells transfected by shRNA aimming at HBx together with independent control series:we should reanimate three kinds of Hepatocellular carcinoma cell(MHCC97-H,HK3,21543),cultivate them in the high glucose DMEM with G418 for some days,then observe the expresion of GFP in cells through inversion fluorescent microscope and initial to estimate the transfection efficiency ,take photographs at last.
2.Detect the extent of HBx gene by RNA interference by semipuanitative RT-PCR : collect three kinds of cell and extract total RNA,add two different pairs of primer and carry out RT-PCR in accordance with illustration,On the basis of endogenous gene strap in the gel electrophoresis,we assessment the tempate quantity and judge the extent of HBx gene by RNA interference.
3.0bserve the influence of cell growth through RNA interference:we should utilize the Cell counting kit-8 and evaluate optical density value ,then we draw the growth curve of three kinds of cell in six days.
4.Detect the diversity of cell cycle by flow cytometry
5.Draw the growth curve of three kinds of cell after add different density of flurouracil ,cisplatin andα-interferon and select the best combination.
6.Detect the cell apoptosis by TUNEL apoptosis detection kit
Result:
1 .There are no difference in the shape and GFP expression intensity in two transfected cell after resuscitation.
2.The result of RT-PCR demonstrate that the HBX mRNA level of 21543 cell down regulate, the HBx mRNA level of HK3 cell is not different with MHCC97-H cell.
3.The growth of 21543 cell are slower than MHCC97-H cell and HK3 cell obviously. The latter two cells are not different notably.
4.The cell cycle of 21543 cell showed that Hepatocellular carcinoma cell through RNA interference targeting to HBx delay to goto S stage,then proliferation activity degrade obviously.
5.Three kinds of cell adding different density of flurouracil ,cisplatin grows slower than the original cell. The growth inhibiting is dependent on the density of drug and the growth inhibiting of 21543 cell is the most obvious.That of three kind of cell adding toα-interferon is not obvious.
6. Flurouracil induce apoptosis in all kinds of cell . The extent of apoptosis in 21543 cell is most obvious.
Conclusion: 1. RNA interference targeting to HBx can supress the growth of hepatocellular carcinoma cell.
2. Hepatocellular carcinoma cell through RNA interference targeting to HBx can intensify chemo-sensitivity.
3. Combination RNA interference targeting to HBx with chemotherapeutics can induced apoptosis in more hepatocellular carcinoma cell.Cell proliferation step down accordingly.
引文
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[57]Hirata A,Ogawa S,Kometani T,et al.ZD1839(Iressa)induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase.Cancer Res,2002,62:2554-2560.
[58]Tsai CM,Chang KT,Li L,et al.Interrelationships between cellular ucleotide excision repair,cisplatin cytotoxicity,HER22/neu gene expression,and epidermal growth factor receptor level in non-small cell lung cancer cells.Jpn J Cancer Res,2000,91:213-222.
[59]Wu Wei-zhong,Sun Hui-chuan,Gao Yan-qin,Wang Lu,Tang Zhao-you,Liu Kang-da.Reconstructing the JAK/STATs signal pathway restored the anti-proliferative response of MHCC-97 on interferon alpha Chin J Hepatol,April2006,Vol14,No.4.
[1]Hammond SM,Bemstein E,Beach D,et al.An RNA-directed nuclease mediates post-transcrip tional gene silencing in Drosophila cells.Nature,2000,404(6775):293-296.
[2]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl To Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature. 2001 May 24;411(6836):494-8
[3] Bernstein E , CaudyAA , Hammond SM , et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , 2001, 409 (6818): 363 - 366.
[4] Elbashir SM , LendeckelW, Tuschl T. RNA interference ismediated by 21 -and 22 - nucleotide RNAs. Genes Dev, 2001,15 (2) :188 - 200.
[5] Elbashir SM , Harborth J , LendeckelW, et al. Dup lexes of 21 -nuckeotide RNAs mediate RNA interference in cultured mammalian cells. Nature , 2001 , 411 (6836): 494 - 498.
[6] Bernstein E , CaudyAA , Hammond SM , et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , 2001, 409 (6818): 363 - 366.
[7] Hannon GJ. RNA interference. Nature, 2002,418 (6894): 244 -251.
[8] Dalmay T , Hamilton A , Rudd S , et al. An RNA - dependent RNA polymerase gene in Arabidop sis is required for posttranscrip tional gene silencing mediated by a transgene but not by a virus. Cell, 2000,101 (5): 543 - 553.
[9]Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA p rimers convertmRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001,107 (3) : 297 - 307.
[10]Ngo H , Tschudi C , Gull K, et al. Double - stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc NatlAcad Sci USA ,1998,95 (25): 14687 -14692.
[11] MontgomeryMK, Xu S , Fire A. RNA as a target of double - stranded RNA mediated genetic interference in Caenorhabditis elegans.Proc Natl Acad Sci USA, 1998,95 (25): 15502 -15507.
[12] Clemens JC , Worby CA , Simonson LeffN , et al. Use of double -stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA, 2000, 97 (12) :6499 - 6503.
[15]Aoki Y,Cioca DP,Oidaira H,et al.RNA interference may be more potent than antisense RNA in human cancer cell lines.Clin Exp Pharmacol Physiol.2003Jan-Feb;30(1-2):96-102.
[16]Steppan CM,Bailey ST,Bhat S,et al.The hormone resistin links obesity to diabetes.Nature,2001,409:307-312
[17]Chan DW,NQ IO1Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells[J]1J Pathol,2006,208(3):3721
[18]Xu Y,Wang YH,Gao JD,Ye J,Zhu HX,Xu NZ,Wang XY,Sun ZT [Suppression of c-mye expression by interference RNA in HepG2 hepatocellular carcinoma eells]Zhonghua Zhong Liu Za Zhi.2004 Aug;26(8):458-60.
[19]Li H,Fu X,Chen Y,Hong Y,Tan Y,Cao H,Wu M,Wang H.Use of adenovirus-delivered siRNA to target oncoprotein p28GANK in hepatocellular carcinoma.Gastroenterology.2005 Jun;128(7):2029-41.
[20]Xue W,Zender L,Miething C,Dickins RA,Hemando E,Krizhanovsky V,Cordon-Cardo C,Lowe SW.Senescence and tumour clearance is triggered by p53restoration in murine liver carcinomas.Nature.2007 Feb 8;445(7128):656-60.
[22]PANG RW,LEE TK,MAN K,et al 1PIN1 expression contributes to hepatic carcinogenesis[J]1J Pathol,2006,210(1):191
[23]Wang S,Lan F,Huang L,Dong L,Zhu Z,Li Z,Xie Y,Fu JSuppression of hLRH-1 mediated by a DNA vector-based RNA interference results in cell cycle arrest and induction of apoptosis in hepatocellular carcinoma cell BEL-7402.Biochem Biophys Res Commun.2005 Aug 5;333(3):917-24.
[24]Cheng SQ,Wang WL,Yan W,Li QL,Wang L,Wang WY Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721.World J Gastroenterol.2005 Feb 7,11(5):756-9
[25]Yan G,Huang AL,Tang N,Zhang BQ,Pu D,Xianh MQ,Lan YH,Wu G.[Inhibition of survivin expression in liver cancer cells by shRNA]Zhonghua Gan Zang Bing Za Zhi.2003 Dec;11(12):712-5
[26]Muse ED,Obici S,Bhanot S,et al.Role of resistin in diet induced hepatic insulin resistance.J Clin Invest,2004,114:232-239
[27] Zhang PH,Zou L,Tu ZG. RNAi-hTERT inhibition hepatocellular carcinoma cell proliferation via decreasing telomerase activity.J Surg Res. 2006 Mar;131(1):143-9. Epub 2005 Nov 18
[28]Lu XD, Qin WX, Pan DN, Li JJ, Wan DF, Wen CJ, Li CJ, Gu JR, Yang SL A DNA vector-based RNAi technology to inhibit the activity of the telomerase of cell line HCCLM3]Zhonghua Yi Xue Za Zhi. 2004 Aug 17;84(16):1381-5.
[29]Zhang PH, Tu ZG, Yang MQ, Huang WF, Zou L, Zhou YL ,Ai Zheng Experimental research of targeting hTERT gene inhibited in hepatocellular carcinoma therapy by RNA interference 2004 Jun;23(6):619-25
[30]Liu QY, Liu ZS, Wu KL, Zhu Y[RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells]Zhonghua Gan Zang Bing Za Zhi. 2005 May;13(5):335-8.
[31]Salvi A, Arici B, De Petro G, Barlati S. Small interfering RNA urokinase silencing inhibits invasion and migration of human hepatocellular carcinoma cells.Mol Cancer Ther. 2004 Jun;3(6):671-8
[32]Cheng J, Huo DH, Kuang DM, Yang J, Zheng L, Zhuang SM. Human macrophages promote the motility and invasiveness of osteopontin-knockdown tumor cells.Cancer Res. 2007 Jun 1;67(11):5141-7
[33]Jia L, Wang S, Cao J, Zhou H, Wei W, Zhang J. siRNA targeted against matrix metalloproteinase 11 inhibits the metastatic capability of murine hepatocarcinoma cell Hca-F to lymph nodes.Int J Biochem Cell Biol. 2007;39(11):2049-62. Epub 2007 Jun 12.
[34]Salvi A, Arici B, Portolani N, Giulini SM, De Petro G, Barlati S. In vitro c-met inhibition by antisense RNA and plasmid-based RNAi down-modulates migration and invasion of hepatocellular carcinoma cells.Int J Oncol. 2007 Aug;31(2):451-60.
[35]Fleischer B, Schulzeberg Kamen H, et allmcl21 is an anti-apop totic factor for human hepatocellular carcinoma[J]1 Int J Oncol,2006,28: 251
[36] Schulzeberg Kamen H, Fleischer B, Schuchmannm, et.all Suppression of mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apop tosis induction[J]1BMC Cancer,2006,6:2321
[37]Lasagna N,Fantappie O,Solazzo M,Morbidelli L,Marchetti S,Cipriani G,Ziche M,Mazzanti R Hepatocyte growth factor and inducible nitric oxide synthase are involved in multidmg resistance-induced angiogenesis in hepatocellular carcinoma cell lines Cancer Res.2006 Mar 1;66(5):2673-82.
[38]Niu J,Li XN,Qian H,Han Z siRNA mediated the type 1 insulin-like growth factor receptor and epidermal growth factor receptor silencing induces chemosensitization of liver cancer cells.J Cancer Res Clin Oncol.2008Apr;134(4):503-13.Epub 2007 Sep 28.
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[59]Wu Wei-zhong,Sun Hui-chuan,Gao Yan-qin,Wang Lu,Tang Zhao-you,Liu Kang-da.Reconstructing the JAK/STATs signal pathway restored the anti-proliferative response of MHCC-97 on interferon alpha Chin J Hepatol,April2006,Vol14,No.4.
[1]Hammond SM,Bemstein E,Beach D,et al.An RNA-directed nuclease mediates post-transcrip tional gene silencing in Drosophila cells.Nature,2000,404(6775):293-296.
[2]Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl To Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature. 2001 May 24;411(6836):494-8
[3] Bernstein E , CaudyAA , Hammond SM , et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , 2001, 409 (6818): 363 - 366.
[4] Elbashir SM , LendeckelW, Tuschl T. RNA interference ismediated by 21 -and 22 - nucleotide RNAs. Genes Dev, 2001,15 (2) :188 - 200.
[5] Elbashir SM , Harborth J , LendeckelW, et al. Dup lexes of 21 -nuckeotide RNAs mediate RNA interference in cultured mammalian cells. Nature , 2001 , 411 (6836): 494 - 498.
[6] Bernstein E , CaudyAA , Hammond SM , et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , 2001, 409 (6818): 363 - 366.
[7] Hannon GJ. RNA interference. Nature, 2002,418 (6894): 244 -251.
[8] Dalmay T , Hamilton A , Rudd S , et al. An RNA - dependent RNA polymerase gene in Arabidop sis is required for posttranscrip tional gene silencing mediated by a transgene but not by a virus. Cell, 2000,101 (5): 543 - 553.
[9]Lipardi C, Wei Q, Paterson BM. RNAi as random degradative PCR: siRNA p rimers convertmRNA into dsRNAs that are degraded to generate new siRNAs. Cell, 2001,107 (3) : 297 - 307.
[10]Ngo H , Tschudi C , Gull K, et al. Double - stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc NatlAcad Sci USA ,1998,95 (25): 14687 -14692.
[11] MontgomeryMK, Xu S , Fire A. RNA as a target of double - stranded RNA mediated genetic interference in Caenorhabditis elegans.Proc Natl Acad Sci USA, 1998,95 (25): 15502 -15507.
[12] Clemens JC , Worby CA , Simonson LeffN , et al. Use of double -stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA, 2000, 97 (12) :6499 - 6503.
[15]Aoki Y,Cioca DP,Oidaira H,et al.RNA interference may be more potent than antisense RNA in human cancer cell lines.Clin Exp Pharmacol Physiol.2003Jan-Feb;30(1-2):96-102.
[16]Steppan CM,Bailey ST,Bhat S,et al.The hormone resistin links obesity to diabetes.Nature,2001,409:307-312
[17]Chan DW,NQ IO1Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells[J]1J Pathol,2006,208(3):3721
[18]Xu Y,Wang YH,Gao JD,Ye J,Zhu HX,Xu NZ,Wang XY,Sun ZT [Suppression of c-mye expression by interference RNA in HepG2 hepatocellular carcinoma eells]Zhonghua Zhong Liu Za Zhi.2004 Aug;26(8):458-60.
[19]Li H,Fu X,Chen Y,Hong Y,Tan Y,Cao H,Wu M,Wang H.Use of adenovirus-delivered siRNA to target oncoprotein p28GANK in hepatocellular carcinoma.Gastroenterology.2005 Jun;128(7):2029-41.
[20]Xue W,Zender L,Miething C,Dickins RA,Hemando E,Krizhanovsky V,Cordon-Cardo C,Lowe SW.Senescence and tumour clearance is triggered by p53restoration in murine liver carcinomas.Nature.2007 Feb 8;445(7128):656-60.
[22]PANG RW,LEE TK,MAN K,et al 1PIN1 expression contributes to hepatic carcinogenesis[J]1J Pathol,2006,210(1):191
[23]Wang S,Lan F,Huang L,Dong L,Zhu Z,Li Z,Xie Y,Fu JSuppression of hLRH-1 mediated by a DNA vector-based RNA interference results in cell cycle arrest and induction of apoptosis in hepatocellular carcinoma cell BEL-7402.Biochem Biophys Res Commun.2005 Aug 5;333(3):917-24.
[24]Cheng SQ,Wang WL,Yan W,Li QL,Wang L,Wang WY Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721.World J Gastroenterol.2005 Feb 7,11(5):756-9
[25]Yan G,Huang AL,Tang N,Zhang BQ,Pu D,Xianh MQ,Lan YH,Wu G.[Inhibition of survivin expression in liver cancer cells by shRNA]Zhonghua Gan Zang Bing Za Zhi.2003 Dec;11(12):712-5
[26]Muse ED,Obici S,Bhanot S,et al.Role of resistin in diet induced hepatic insulin resistance.J Clin Invest,2004,114:232-239
[27] Zhang PH,Zou L,Tu ZG. RNAi-hTERT inhibition hepatocellular carcinoma cell proliferation via decreasing telomerase activity.J Surg Res. 2006 Mar;131(1):143-9. Epub 2005 Nov 18
[28]Lu XD, Qin WX, Pan DN, Li JJ, Wan DF, Wen CJ, Li CJ, Gu JR, Yang SL A DNA vector-based RNAi technology to inhibit the activity of the telomerase of cell line HCCLM3]Zhonghua Yi Xue Za Zhi. 2004 Aug 17;84(16):1381-5.
[29]Zhang PH, Tu ZG, Yang MQ, Huang WF, Zou L, Zhou YL ,Ai Zheng Experimental research of targeting hTERT gene inhibited in hepatocellular carcinoma therapy by RNA interference 2004 Jun;23(6):619-25
[30]Liu QY, Liu ZS, Wu KL, Zhu Y[RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells]Zhonghua Gan Zang Bing Za Zhi. 2005 May;13(5):335-8.
[31]Salvi A, Arici B, De Petro G, Barlati S. Small interfering RNA urokinase silencing inhibits invasion and migration of human hepatocellular carcinoma cells.Mol Cancer Ther. 2004 Jun;3(6):671-8
[32]Cheng J, Huo DH, Kuang DM, Yang J, Zheng L, Zhuang SM. Human macrophages promote the motility and invasiveness of osteopontin-knockdown tumor cells.Cancer Res. 2007 Jun 1;67(11):5141-7
[33]Jia L, Wang S, Cao J, Zhou H, Wei W, Zhang J. siRNA targeted against matrix metalloproteinase 11 inhibits the metastatic capability of murine hepatocarcinoma cell Hca-F to lymph nodes.Int J Biochem Cell Biol. 2007;39(11):2049-62. Epub 2007 Jun 12.
[34]Salvi A, Arici B, Portolani N, Giulini SM, De Petro G, Barlati S. In vitro c-met inhibition by antisense RNA and plasmid-based RNAi down-modulates migration and invasion of hepatocellular carcinoma cells.Int J Oncol. 2007 Aug;31(2):451-60.
[35]Fleischer B, Schulzeberg Kamen H, et allmcl21 is an anti-apop totic factor for human hepatocellular carcinoma[J]1 Int J Oncol,2006,28: 251
[36] Schulzeberg Kamen H, Fleischer B, Schuchmannm, et.all Suppression of mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apop tosis induction[J]1BMC Cancer,2006,6:2321
[37]Lasagna N,Fantappie O,Solazzo M,Morbidelli L,Marchetti S,Cipriani G,Ziche M,Mazzanti R Hepatocyte growth factor and inducible nitric oxide synthase are involved in multidmg resistance-induced angiogenesis in hepatocellular carcinoma cell lines Cancer Res.2006 Mar 1;66(5):2673-82.
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