慢病毒载体介导siRNA沉默Id-1抑制裸鼠人肝癌移植瘤生长的实验研究
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摘要
目的:
通过构建慢病毒表达载体介导siRNA沉默Id-1,观察其对人肝癌HepG2裸小鼠皮下移植瘤生长的抑制作用及其对ERK1/2信号通路的影响,探讨其参与原发性肝细胞癌增殖的可能机制,为寻找原发性肝细胞癌新的治疗靶点提供实验依据。
方法:
构建设计以Id-1基因为靶序列的5个慢病毒表达载体介导的shRNA,生产收集慢病毒上清液(3×106TU/ml),转染肝癌HepG2细胞系,筛选稳定沉默Id-1基因表达的细胞系,RT-QPCR鉴定筛选最佳沉默效果的细胞系。18只BALA/c nu/nu系雄性裸小鼠随机分为3组,即转染实验组(Id-1-RNAi-Lentivirus)、阴性对照组(GFP-lentivirus)、空白对照组(正常HepG2细胞),每组6只。分别取细胞浓度为5×106/ml的干扰效率最佳的稳定转染细胞系、稳定转染空载体病毒细胞系、正常培养未干扰的HepG2细胞系悬液各0.2ml,在裸鼠右腋皮下注射,连续观察28天,期间每周测量肿瘤体积、裸鼠体重,绘制肿瘤生长曲线,28天后处死裸鼠,制作肿瘤组织标本,行常规病理检查,半定量RT-PCR、Western bolt检测Id-1、ERK1/2、p-ERK1/2mRNA及蛋白水平的表达。
结果:
倒置荧光显微镜观察转染前后细胞形态学变化不明显;RT-QPCR检测sh31肝癌HepG2稳定细胞系Id-1沉默效果最佳,与稳定转染空载体病毒细胞系相比降低80%以上,与未干扰的HepG2细胞相比降低了60%左右;裸鼠成瘤3-5天后,可在皮下触到直径约3mm-4mm肿瘤结节,成瘤率100%,转染实验组瘤体增长缓慢,阴性对照组及空白对照组瘤体增长迅速;病理组织学显示:肉眼观察,实验组的裸鼠皮下肿瘤体积明显较对照组小。镜下观察,空白对照组及阴性对照组肿瘤细胞密集排列,片状坏死区面积较小,核大深染,可见核分裂象,核固缩较少。实验组肿瘤细胞呈大片状坏死,空泡性变,多数细胞核完全溶解,细胞结构消失,呈均质、红染表现,可见多数凋亡细胞;RT-PCR显示转染实验组Id-1、ERK1/2、p-ERK1/2mRNA表达水平明显降低,与对照组相比差别有统计学意义(P<0.05);Western bolt显示转染实验组Id-1、p-ERK1/2蛋白表达水平亦明显下调,与对照组相比差别有统计学意义(P<0.05),而ERK1/2蛋白表达变化不明显(P>0.05)。
结论:
通过构建携带Id-1基因特异性siRNA的慢病毒表达载体,并转染肝癌HepG2细胞系稳定沉默Id-1基因的表达及采用肝癌HepG2细胞系在裸小鼠皮下注射移植成瘤建立肝癌模型的方法是有效和可行的。下调Id-1表达后,可以有效地抑制裸鼠皮下移植瘤的生长,抑制肿瘤细胞的增殖,且ERK1/2的活性形式p-ERK1/2也随之表达减少,推测Id-1可能通过调节ERK1/2MAPK信号通路参与肝癌的发生发展。Id-1在肝细胞癌中的高表达及下调Id-1后可有效抑制肝细胞癌增殖,提示抑制靶基因Id-1的表达可能为肝癌的基因治疗提供一种新的治疗途径。
Objective:
To construct a lentiviral vector carrying siRNA to silence Id-1, and observe its inhibition of human hepatocellular carcinoma HepG2 of subcutaneous tumor in nude mice and its effect on ERK1/ 2 signaling pathway.Investigate a possible mechanism of their participation in the proliferation of HCC, and provide experimental evidence for a new therapeutic targets in primary hepatocellular carcinoma.
Methods:
Using Id-1 gene as target sequence, 5 lentiviral vector mediated shRNA were constructed, and collected lentiviral supernatants(3×106TU/ml),which then were transfected into HepG2 cell line.After screening the cell lines stable silencing the expression of Id-1,the cell lines with best silencing effect were iderntified by RT-QPCR.18 BALA/c nu/nu Male nude mice were randomly divided into 3 groups with equal number:the transfected experimental group(treated with Id-1-RNAi-Lentiv -irus),the negative control group(treated with GFP-lentivirus),and the control group(treated with PBS). subcutaneous injection in nude mice’s right axillary with 0.2ml the best interferential efficiency cell lines,vector virus treated cell lines and nomal HepG2 cell lines, The concentration of all these cell lines were 5×106/ml. Continuous observation for 28 days, during which time the tumor volume and the weight of nude mice were measured every week, and the tumor growth curve was mapped.After 28 days,mice were killed and make the tumor specimens and perform routine pathological examination, moreover, the expression of Id-1、ERK1/2、p-ERK1/2 in mRNA and protein level were examined by Semi-quantitative RT-PCR and Western bolt,respectively.
Resulte:
Cell morphology changes not evident before and after transfection observed by inverted fluorescence microscope. RT-QPCR identified that sh31 liver cancer HepG2 stable cell lines have the best silence effect, reducing the expression of Id-1 more than 80% compared to stable empty virus vector cell lines while about 60% compared to normal HepG2 cell lines. Three to five days after tumor formation in nude mice, tumor nodules whose diameter is about 3mm-4mm subcutaneously can be touched and the percentage of tumorigenesis is 100%.Tumor grow much more sharply in negative control group and control group than in transfected experimental group and gradually increase in negative control group and control group with time. Pathology histology display that by naked eye observation, the size of tumor in transfected experimental group is obviously smaller than that in control group. however,in microscope, tumor cells of control group arrange densely and grow vigorously with various size and shape.In addition, these cells has little karyopyknosis and small flake necrosis areas meanwhile large darkly stained nuclear.Conversely,in transfected experimental group ,tumor cells are large necrosis and vaculation with homogeneous and red dye performance.Besides,most of the nucleus is completely dissolved and cell structure disappeared,while most cells are apoptosis and appear cytoplasmic concentration, condensation nuclei stained.RT-PCR results display that the mRNA expression levels of Id-1, ERK1/2, p-ERK1/2 in transfected experimental group are obviously decreased while the difference to those in control group are statistical significance(P<0.05).Western bolt results show that the protein expression levels of Id-1, ERK1/2 in transfected experimental group are also obviously decreased,the difference to those in control group are also statistical significance ( P <0.05).However,the protein expression level of ERK1/2 protein expression does not change significantly(P>0.05).
Conclusion:
Constructed a lentiviral vector carrying specific siRNA for Id-1 , then transfected it into the hepatoma cell line HepG2 to silence the expression of Id-1 gene stably and the way to establish a liver tumor model by transplanting subcutaneously in nude mice with hepatocellular carcinoma HepG2 cell line is effective and feasible. the growth of nude mice and the proliferation of tumor cell have been inhibited when Id-1 have been down-regulated, in adddtion,p-ERK1/2,the activated format of ERK1/2 are also decreased,which indicated that Id-1 may influence the development of liver cancer by regulate ERK1/2MAPK signaling pathway.The fact that Id-1 is highly expressed in hepatocellular carcinoma and down-regulation of the Id-1 could inhibit the proliferation of hepatocellular carcinoma suggest that inhibiting the expression of Id-1 may become a new treatment for liver cancer’s gene therapy.
通过构建慢病毒表达载体介导siRNA沉默Id-1,观察其对人肝癌HepG2裸小鼠皮下移植瘤生长的抑制作用及其对ERK1/2信号通路的影响,探讨其参与原发性肝细胞癌增殖的可能机制,为寻找原发性肝细胞癌新的治疗靶点提供实验依据。
方法:
构建设计以Id-1基因为靶序列的5个慢病毒表达载体介导的shRNA,生产收集慢病毒上清液(3×106TU/ml),转染肝癌HepG2细胞系,筛选稳定沉默Id-1基因表达的细胞系,RT-QPCR鉴定筛选最佳沉默效果的细胞系。18只BALA/c nu/nu系雄性裸小鼠随机分为3组,即转染实验组(Id-1-RNAi-Lentivirus)、阴性对照组(GFP-lentivirus)、空白对照组(正常HepG2细胞),每组6只。分别取细胞浓度为5×106/ml的干扰效率最佳的稳定转染细胞系、稳定转染空载体病毒细胞系、正常培养未干扰的HepG2细胞系悬液各0.2ml,在裸鼠右腋皮下注射,连续观察28天,期间每周测量肿瘤体积、裸鼠体重,绘制肿瘤生长曲线,28天后处死裸鼠,制作肿瘤组织标本,行常规病理检查,半定量RT-PCR、Western bolt检测Id-1、ERK1/2、p-ERK1/2mRNA及蛋白水平的表达。
结果:
倒置荧光显微镜观察转染前后细胞形态学变化不明显;RT-QPCR检测sh31肝癌HepG2稳定细胞系Id-1沉默效果最佳,与稳定转染空载体病毒细胞系相比降低80%以上,与未干扰的HepG2细胞相比降低了60%左右;裸鼠成瘤3-5天后,可在皮下触到直径约3mm-4mm肿瘤结节,成瘤率100%,转染实验组瘤体增长缓慢,阴性对照组及空白对照组瘤体增长迅速;病理组织学显示:肉眼观察,实验组的裸鼠皮下肿瘤体积明显较对照组小。镜下观察,空白对照组及阴性对照组肿瘤细胞密集排列,片状坏死区面积较小,核大深染,可见核分裂象,核固缩较少。实验组肿瘤细胞呈大片状坏死,空泡性变,多数细胞核完全溶解,细胞结构消失,呈均质、红染表现,可见多数凋亡细胞;RT-PCR显示转染实验组Id-1、ERK1/2、p-ERK1/2mRNA表达水平明显降低,与对照组相比差别有统计学意义(P<0.05);Western bolt显示转染实验组Id-1、p-ERK1/2蛋白表达水平亦明显下调,与对照组相比差别有统计学意义(P<0.05),而ERK1/2蛋白表达变化不明显(P>0.05)。
结论:
通过构建携带Id-1基因特异性siRNA的慢病毒表达载体,并转染肝癌HepG2细胞系稳定沉默Id-1基因的表达及采用肝癌HepG2细胞系在裸小鼠皮下注射移植成瘤建立肝癌模型的方法是有效和可行的。下调Id-1表达后,可以有效地抑制裸鼠皮下移植瘤的生长,抑制肿瘤细胞的增殖,且ERK1/2的活性形式p-ERK1/2也随之表达减少,推测Id-1可能通过调节ERK1/2MAPK信号通路参与肝癌的发生发展。Id-1在肝细胞癌中的高表达及下调Id-1后可有效抑制肝细胞癌增殖,提示抑制靶基因Id-1的表达可能为肝癌的基因治疗提供一种新的治疗途径。
Objective:
To construct a lentiviral vector carrying siRNA to silence Id-1, and observe its inhibition of human hepatocellular carcinoma HepG2 of subcutaneous tumor in nude mice and its effect on ERK1/ 2 signaling pathway.Investigate a possible mechanism of their participation in the proliferation of HCC, and provide experimental evidence for a new therapeutic targets in primary hepatocellular carcinoma.
Methods:
Using Id-1 gene as target sequence, 5 lentiviral vector mediated shRNA were constructed, and collected lentiviral supernatants(3×106TU/ml),which then were transfected into HepG2 cell line.After screening the cell lines stable silencing the expression of Id-1,the cell lines with best silencing effect were iderntified by RT-QPCR.18 BALA/c nu/nu Male nude mice were randomly divided into 3 groups with equal number:the transfected experimental group(treated with Id-1-RNAi-Lentiv -irus),the negative control group(treated with GFP-lentivirus),and the control group(treated with PBS). subcutaneous injection in nude mice’s right axillary with 0.2ml the best interferential efficiency cell lines,vector virus treated cell lines and nomal HepG2 cell lines, The concentration of all these cell lines were 5×106/ml. Continuous observation for 28 days, during which time the tumor volume and the weight of nude mice were measured every week, and the tumor growth curve was mapped.After 28 days,mice were killed and make the tumor specimens and perform routine pathological examination, moreover, the expression of Id-1、ERK1/2、p-ERK1/2 in mRNA and protein level were examined by Semi-quantitative RT-PCR and Western bolt,respectively.
Resulte:
Cell morphology changes not evident before and after transfection observed by inverted fluorescence microscope. RT-QPCR identified that sh31 liver cancer HepG2 stable cell lines have the best silence effect, reducing the expression of Id-1 more than 80% compared to stable empty virus vector cell lines while about 60% compared to normal HepG2 cell lines. Three to five days after tumor formation in nude mice, tumor nodules whose diameter is about 3mm-4mm subcutaneously can be touched and the percentage of tumorigenesis is 100%.Tumor grow much more sharply in negative control group and control group than in transfected experimental group and gradually increase in negative control group and control group with time. Pathology histology display that by naked eye observation, the size of tumor in transfected experimental group is obviously smaller than that in control group. however,in microscope, tumor cells of control group arrange densely and grow vigorously with various size and shape.In addition, these cells has little karyopyknosis and small flake necrosis areas meanwhile large darkly stained nuclear.Conversely,in transfected experimental group ,tumor cells are large necrosis and vaculation with homogeneous and red dye performance.Besides,most of the nucleus is completely dissolved and cell structure disappeared,while most cells are apoptosis and appear cytoplasmic concentration, condensation nuclei stained.RT-PCR results display that the mRNA expression levels of Id-1, ERK1/2, p-ERK1/2 in transfected experimental group are obviously decreased while the difference to those in control group are statistical significance(P<0.05).Western bolt results show that the protein expression levels of Id-1, ERK1/2 in transfected experimental group are also obviously decreased,the difference to those in control group are also statistical significance ( P <0.05).However,the protein expression level of ERK1/2 protein expression does not change significantly(P>0.05).
Conclusion:
Constructed a lentiviral vector carrying specific siRNA for Id-1 , then transfected it into the hepatoma cell line HepG2 to silence the expression of Id-1 gene stably and the way to establish a liver tumor model by transplanting subcutaneously in nude mice with hepatocellular carcinoma HepG2 cell line is effective and feasible. the growth of nude mice and the proliferation of tumor cell have been inhibited when Id-1 have been down-regulated, in adddtion,p-ERK1/2,the activated format of ERK1/2 are also decreased,which indicated that Id-1 may influence the development of liver cancer by regulate ERK1/2MAPK signaling pathway.The fact that Id-1 is highly expressed in hepatocellular carcinoma and down-regulation of the Id-1 could inhibit the proliferation of hepatocellular carcinoma suggest that inhibiting the expression of Id-1 may become a new treatment for liver cancer’s gene therapy.
引文
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[52]Eid MA,Kumar MV,Iczkowski KA,et al.Expression of early growth response genes in human prostate cancer[J].Cancer Res,1998, 58 (11): 2461-2468.
[53]Albanell J,Codony-Servat J, Rojo F, et al. Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments[J].Cancer Res, 2001, 61, (17): 6500-6510.
[1]Yokota Y,Mori S.Role of Id family proteins in growth control[J].J Cell Physiol 2002,190(1):l21-128.
[2]Ruzinova MB,Benezra R. Id proteins in development, cell cycle and cancer[J]. Trends Cell Biol,2003,13(8): 410-418.
[3]Zhao ZR,Zhang ZY,Zhang H,et al. Over expression of Id-1 protein is a marker in colorectal cancer progression[J]. Oncol Rep,2008, 19(2):419-424.
[4]Hui CM, Cheung PY, Ling MT, et al. Id-1 promotes proliferation of p53-deficient esophageal cancer cells[J]. Int J Cancer, 2006, 119(3):508-514.
[5]Lee TK,Poon RTP,Yuen AP, et al. Regulation of angiogenesis by Id-1 through hypoxia-inducible factor-1a-mediated vascular endothelial growth factor up-regulat -ion in hepatocellular carcinoma[J].Clin Cancer Res,2006,12(23):6910–6919.
[6]Di K,Wong YC,Wang X.Id-1 promotes TGF-β1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells[J].Exp Cell Res,2007,313(19):3983–3999.
[7]Zhang X,Ling MT,Wong YC, et al. Evidence of a novel antiapoptotic factor: role of inhibitor of differentiation or DNA binding (Id-1) in anticancer drug-induced apoptosis[J]. Cancer Sci, 2007,98(3):308–314.
[8]Schoppmann SF,Schindl M,Bayer G,et a1.Over expression of Id-1 is associated with poor clinical out come in node negative breast caner[J]. Int J Cancer,2003,104(6):67 7-682.
[9]Ling MT,Wang X,Ouyang XS,et al.Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth[J].Oncong-ne, 2002,21(55):8498-8505.
[10]刘豫瑞,方明霞,曾达武.Id-1在肝细胞癌中的表达及其临床意义[J].福建医科大学学报,2009,43(1):32-36.
[11]Ouyang XS,Wang X,Ling MT,et al.Id-1 stimulates serum independent prosrate cancer cell proliferation through inactication of p16INK4a/pRB pathway[J]. Carcino -genesis,2002,23(5):721-725.
[12]Zhang X,Ling MT,Wong YC,et al.Id-1 stimulates celll proliferation through activation of EGFR in ovarian cancer cells[J].Br J Cancer,2004,91(12):2042-2047.
[13]Cheung HW,Ling MT,Tsao SW,et al.Id-1-induced Raf/MEK pathway activation is essential for its protective role against taxol-induced apoptosis in nasopharyngeal carcinoma cells[J].carrcinogenesis,2004,25(6):881-887.
[14]Ling MT,Wang X,Ouyang X,et a1.Id-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells[J]. Oncogene, 2003,22(29):4498-4508.
[15] Lin JC,Chang SY,Hsieh DS,et a1.Modulation of mitogen-activated protein kinase cascades by differentiation-1 protein: acquired drug resistance of hormone independent prostate cancer cells[J].J Urol,2005,174(5):2022-2026.
[16]Ling MT,Kwok WK,Fung MK,et a1.Proteasome mediated degradation of Id-1 is associated with TNFa-induced apoptosis in prostate cancercells[J]. Carcinogenesis, 2006,27(2):205-215.
[17]Han S,Guo C,Hong L,et a1.Expression and significances of Id1 helix-loop-helix protein overexpression in gastric cancer[J].Cancer Lett,2004,216(1):63-71.
[18]Ding Y,Wang G,Ling MT,et a1.Signitleanee of Id-l upregulation and its association with EGFR in bladder cancer cell invasion[J].Int J Oncol,2006,28(4):847—854.
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[20]Fong S,Itaham Y,Sumida T,et al.ld-l as a molecular target in therapy for breast cancer cell invasion and metastasis[J]. Proc Natl Acad Sci U S A,2003, 100(23):13543-13548.
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[24]Sakurai D,Tsuchiya N,Yamaguchi A,etal.Endothelial Cells Angiogenic Processes of HumanFactor-Induced Activation and Vascular Endothelial Growth Binding/Differentiation in the Crucial Role of Inhibitor of DNA[J].Immunol, 2004,173(9):5801-5809.
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[1]Yokota Y,Mori S.Role of Id family proteins in growth control[J].J Cell Physiol 2002,190(1):l21-128.
[2]Ruzinova MB,Benezra R. Id proteins in development, cell cycle and cancer[J]. Trends Cell Biol,2003,13(8): 410-418.
[3]Zhao ZR,Zhang ZY,Zhang H,et al. Over expression of Id-1 protein is a marker in colorectal cancer progression[J]. Oncol Rep,2008, 19(2):419-424.
[4]Hui CM, Cheung PY, Ling MT, et al. Id-1 promotes proliferation of p53-deficient esophageal cancer cells[J]. Int J Cancer, 2006, 119(3):508-514.
[5]Lee TK,Poon RTP,Yuen AP, et al. Regulation of angiogenesis by Id-1 through hypoxia-inducible factor-1a-mediated vascular endothelial growth factor up-regulat -ion in hepatocellular carcinoma[J].Clin Cancer Res,2006,12(23):6910–6919.
[6]Di K,Wong YC,Wang X.Id-1 promotes TGF-β1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells[J].Exp Cell Res,2007,313(19):3983–3999.
[7]Zhang X,Ling MT,Wong YC, et al. Evidence of a novel antiapoptotic factor: role of inhibitor of differentiation or DNA binding (Id-1) in anticancer drug-induced apoptosis[J]. Cancer Sci, 2007,98(3):308–314.
[8]Schoppmann SF,Schindl M,Bayer G,et a1.Over expression of Id-1 is associated with poor clinical out come in node negative breast caner[J]. Int J Cancer,2003,104(6):67 7-682.
[9]Ling MT,Wang X,Ouyang XS,et al.Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth[J].Oncong-ne, 2002,21(55):8498-8505.
[10]刘豫瑞,方明霞,曾达武.Id-1在肝细胞癌中的表达及其临床意义[J].福建医科大学学报,2009,43(1):32-36.
[11]Ouyang XS,Wang X,Ling MT,et al.Id-1 stimulates serum independent prosrate cancer cell proliferation through inactication of p16INK4a/pRB pathway[J]. Carcino -genesis,2002,23(5):721-725.
[12]Zhang X,Ling MT,Wong YC,et al.Id-1 stimulates celll proliferation through activation of EGFR in ovarian cancer cells[J].Br J Cancer,2004,91(12):2042-2047.
[13]Cheung HW,Ling MT,Tsao SW,et al.Id-1-induced Raf/MEK pathway activation is essential for its protective role against taxol-induced apoptosis in nasopharyngeal carcinoma cells[J].carrcinogenesis,2004,25(6):881-887.
[14]Ling MT,Wang X,Ouyang X,et a1.Id-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells[J]. Oncogene, 2003,22(29):4498-4508.
[15] Lin JC,Chang SY,Hsieh DS,et a1.Modulation of mitogen-activated protein kinase cascades by differentiation-1 protein: acquired drug resistance of hormone independent prostate cancer cells[J].J Urol,2005,174(5):2022-2026.
[16]Ling MT,Kwok WK,Fung MK,et a1.Proteasome mediated degradation of Id-1 is associated with TNFa-induced apoptosis in prostate cancercells[J]. Carcinogenesis, 2006,27(2):205-215.
[17]Han S,Guo C,Hong L,et a1.Expression and significances of Id1 helix-loop-helix protein overexpression in gastric cancer[J].Cancer Lett,2004,216(1):63-71.
[18]Ding Y,Wang G,Ling MT,et a1.Signitleanee of Id-l upregulation and its association with EGFR in bladder cancer cell invasion[J].Int J Oncol,2006,28(4):847—854.
[19]Lin CQ,Sing HJ,Murata K,et a1.A Role for Id-1 in the Aggressive Phenotype and Steroid Hormone Response of Human Breast Cancer Cells[J].Cancer Res,2000,60(5):1332-1340.
[20]Fong S,Itaham Y,Sumida T,et al.ld-l as a molecular target in therapy for breast cancer cell invasion and metastasis[J]. Proc Natl Acad Sci U S A,2003, 100(23):13543-13548.
[21]Amold JM,Mok SC,Purdie D,et al.Decreased expression of the Id3 gene at 1p36.1 in cvarian adenocarcinomas[J].Br J Cancer,2001,84(3):352-359.
[22]Ling MT,Lau TC,Zhou C,et a1.Overexpression of Id-1 in prostate cancer cells promotes angiogenesis through the activation of vascular endothelial growth factor (VEGF)[J].carcinogenesis,2005,26(10):1668-l676.
[23]Lee KT,Lee YW,Lee JK,et a1.Over expression of Id-l is signific antly associated with tumour angiogenesis in human pancreas cancers [J].Br J Cancer,2004,90(6):1198-1203.
[24]Sakurai D,Tsuchiya N,Yamaguchi A,etal.Endothelial Cells Angiogenic Processes of HumanFactor-Induced Activation and Vascular Endothelial Growth Binding/Differentiation in the Crucial Role of Inhibitor of DNA[J].Immunol, 2004,173(9):5801-5809.
[25]Straume 0,Akslen LA.Strong expression of Id1protein is associated with decreased survival,increased expression of ephrin-A1/Epha2,and reduced thromospondin-1 inalignant melanoma[].Br J Cancer,2005,93(8):933-938.
[26]Lydenn D,Young AZ,zagzag D,et al. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts[J].Nature,1999,401(675 4):670-677.