IA型PI3K/Akt信号通路在胃腺癌中表达调控机制的实验研究
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摘要
胃癌是消化系统最常见的恶性肿瘤,死亡率居消化道各类癌症死亡率之首。研究其发病机理、探索有效治疗方法一直是医学研究最重要的课题之一。随着肿瘤生物学和分子遗传学理论与技术的发展,目前已认识到,胃癌是一种多基因异常疾病,和人体其他部位的肿瘤一样,胃癌发生发展主要是原癌基因的激活与抑癌基因的失活并由此产生一系列基因异常和动态演进的结果,因此寻找与胃癌发病相关的基因,深入了解胃癌的分子病理,在此基础上开拓胃癌治疗的新策略和靶点,成为肿瘤研究领域的一项重要内容。
IA型PI3K/Akt通路与肿瘤的发生发展密切相关。该通路调节肿瘤细胞的增殖和存活,其活性增加不仅能导致细胞恶性转化,而且与肿瘤细胞的迁移、黏附、肿瘤血管生成以及细胞外基质的降解等相关。本课题重点研究了胃腺癌中IA型PI3K/Akt通路的异常表达,并应用RNA干涉技术干预该通路中PI3Kp85α的异常激活,观察干预PI3Kp85α后对胃腺癌恶性表型的影响及对裸鼠皮下SGC7901胃腺癌模型的治疗效果。课题分三部分进行:
第一部分应用组织芯片技术结合免疫组化检测IA型PI3K/Akt通路主要成员PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中的表达情况。
第二部分在体外应用RNA干涉技术以oligofectamine介导siRNA靶向PI3Kp85α转染人胃腺癌细胞系SGC7901后,应用realtime PCR检测目的基因PI3Kp85α的敲低情况,并用Western blot和免疫荧光技术检测了RNAi敲低PI3Kp85α的表达后,IA型PI3K/Akt通路主要成员PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2的表达变化:用MTT、annexin V标记、流式细胞术分析了转染前后细胞增殖、凋亡、细胞周期分布的变化:“划痕”法迁移实验和2-D Matrigel法研究细胞运动迁移能力:用Transwell和3-D Mattigel法分析转染前后细胞侵袭能力的变化。
第三部分建立裸鼠胃腺癌皮下动物模型,每4天瘤内注射oligofectamine和siRNA的混合物一次并测量肿瘤体积,动态观察肿瘤生长情况,治疗32天后切除肿瘤。用免疫组化法检测RNA干涉技术敲低SGC7901裸鼠胃腺癌PI3Kp85α的表达后该通路主要成员的表达变化,并应用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。
结果:
第一部分组织芯片结合免疫组化技术显示IA型PI3K/Akt通路中PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中均呈高表达,其表达水平随肿瘤的演进程度及恶性级别增高而增强。
第二部分Realtime PCR结果提示,转染PI3Kp85αsiRNA后可明显敲低染PI3Kp85αmRNA的表达。Western blot和免疫荧光检测发现除PI3Kp85α的蛋白明显敲低外,IA型PI3K/Akt通路中pAkt、Ki67、Bcl-2、MMP-2的表达水平均明显降低。MTT结果显示PI3Kp85αsiRNA组与转染nonsense siRNA组及对照组相比细胞增殖率明显受抑(P＜0.01);Annexin V检测显示PI3Kp85αsiRNA组细胞凋亡率明显升高,进一步应用流式细胞术检测细胞周期分布发现S期细胞比例降低,细胞阻滞于G0/G1期;“划痕”法迁移实验和2-D Matrigel法显示PI3Kp85αsiRNA组较nonsense siRNA组和对照组的细胞的运动迁移能力降低(P＜0.01);Transwell和3-D Matrigel法显示其侵袭能力减弱(P＜0.01)。
第三部分成功建立SGC7901胃腺癌细胞系裸鼠皮下肿瘤后,每4天测量一次肿瘤体积,并在肿瘤局部多点注射siRNA和oligofectamine混合物,发现和对照组相比,PI3Kp85αsiRNA治疗组肿瘤生长缓慢,从治疗后的第20天起,肿瘤体积出现明显差别,这种差别在观察末期(32天)最显著。应用免疫组化法检测肿瘤标本,发现PI3Kp85α的蛋白明显敲低的同时,IA型PI3K/Akt通路中pAkt、Ki67、Bcl-2、MMP-2的表达水平均明显降低。应用TUNEL法检测原位细胞凋亡发现PI3Kp85α治疗组凋亡细胞明显增加。体内和体外实验的结果是一致的。
结论:
1.免疫组化显示PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中都有过表达,表明IA型PI3K/Akt信号通路在胃腺癌中是激活的,从而导致细胞增殖和侵袭。其中各种蛋白的表达相互关联也验证了这一点。
2.应用RNA干涉技术以Oligofectamine介导siRNA靶向PI3Kp85α可有效敲低这个基因在人胃腺癌SGC7901细胞内的表达,同时抑制IA型PI3K/Akt通路的活性,从而抑制了细胞的增殖和侵袭,并诱发细胞凋亡。
3.裸鼠皮下胃腺癌模型应用oligofectamine介导的PI3Kp85αsiRNA治疗,同样可有效敲低PI3Kp85α的表达,抑制IA型PI3K/Akt通路的活性,从而抑制肿瘤的生长,诱导细胞凋亡,与体外试验结果一致。
4.通过RNA干涉技术敲低PI3Kp85α的表达对胃腺癌的治疗是有效的。PI3Kp85α可成为胃腺癌基因治疗的候选基因,具有潜在的临床应用前景。
Gastric carcinoma is the most common and incurable digestive systerm tumor,which mortality is the highest.Studying its pathogenesis and trying to find more effective therapeutic modalities are the major research projects in medical field. Gastric carcinoma,just like tumors in other sites of the body,are involved in the processes of uncontrolled cell proliferation,cell dedifferentiation and dysregulation of cell apoptosis.With the development of molecular biology and molecular genetics,it has been found that some of the important oncogenes and tumor suppressor genes are related to the genesis of gastric carcinoma.However,up to date,it is still not clear which are the primary initiating molecular events and which are the secondary promoting molecular events.Therefore,seeking more genes associated with the genesis of gastric carcinoma,a comprehensive understanding of the molecular pathology of gastric carcinoma,and optimizing treatment strategies and developing novel therapeutic approaches for gastric carcinoma on this basis,have become an important research project for carcinoma.
IA Type PI3K/Akt signaling pathway which regulates the proliferation and survival of tumor cells is closely related to the genesis and development of gastric adenocarcinoma.Its activity abnormality not only can induce cells malignant transfor -mation,but also influence the migration and adhesion of tumor cells,vascularization and degradation of extracellular matrix.The present study focused on the abnormal IA Type PI3K/Akt signaling pathway activity in gastric adenocarcinoma and siRNA targeting PI3Kp85αwas used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells and therapeutic efficacy of nude mice subcutaneous SGC7901 gastric adenocarcinoma model.The study was divided into 3 parts.
In the first part,Using tissue microarray techniques and immunohistochemistry assay to survey the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway during gastric adenocarcinoma malignant progression.
In the second part,RNAi technology was used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells,siRNA targeting PI3Kp85αwas transfected into SGC7901 cells mediated by oligofectamine. PI3Kp85αmRNA expression were detected by realtime PCR after transfection.The expression of PI3Kp85αand other main downstream members including pAkt,Ki67, bcl-2,MMP-2 in IA type PI3K/Akt pathway was also studied by Western blotting and immunofluorescence staining after transfection.The phenotypic change of SGC7901 cells including proliferation,apoptosis and cell cycle after RNAi trasfection was studied by MTT assay,annexin V staining,flow cytometry;their motion and migra -tion ability was studied by scratch assay and 2-D Matrigel assay;the invasion ability was detected by Transwell analysis and 3-D Matrigel assay.
In the third part,subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice.Every 4 days the mixture of oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 32~(nd)day after the first injection,tumors were resected.The expression of PI3Kp85α,pAkt,Ki67,Bcl-2, MMP-2 in IA type PI3K/Akt signaling pathway were studied by immunohistochemis -try.Apoptosis in tumors were detected by TUNEL method.
Results:
The first part:The expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway were overexpressed during gastric adenocarcinoma malignant progression.
The second part:SGC7901 cells were transfected with siRNA targeting PI3Kp85αmediated by oligofectamine in vitro.SGC7901 mRNA expression were obviously knocked down after transfection with siRNA.SGC7901 ceils transfected with siRNA targeting PI3Kp85αshowed lowering proliferation activity by MTT, while the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 were downregulated by Western blot analysis and immunohistochemical staining.The transfected cells had higher apotosis rate by annexin V staining and most cells arresting in the Go/G1phase by flow cytometry.The migration and invasive ability was attenuated by scratch assay,2-D Matrigel assay,Transwell analysis and 3-D Matrigel assay.
The third part:Subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice for in vivo study.As compared with the control group,the tumors in mice treated with siRNA targeting PI3Kp85αgrew slowly and the different ce of tumor volumes became significant since the 20th day after the first time of siRNA therapy until the 32~(nd)day tumors were resected.PI3Kp85α,pAkt,Ki67, Bcl-2,MMP-2's expression were downregulated by immunohistochemistry. Meanwhile,cell apoptosis was increased by TUNEL method.
Conclusion:
1.PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2's overexpression can lead to IA type PI3K/Akt signaling pathway activation in gastric adenocarcinoma and result in cell proliferation and invasion.Meanwhile,the several proteins in this signaling pathway links to each other and maybe they are modulated with each other.
2.Using RNAi technology,siRNA targeting PI3Kp85αmediated by oligofectamine efficiently knocks down the expression of PI3Kp85αin human gastric adenocarcinoma SGC7901 cells,inhibits activation of IA type PI3K/Akt signaling activity,results in decrease of cell proliferation activity and attenuateion of invasive and migration ability,and induces apoptosis.
3.The established subcutaneous SGC7901 gastric adenocarcinoma models in nude mice are treated with siRNA targeting PI3Kp85α.The tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study
4.Using RNAi technology to knock down the expression of PI3Kp85αin gastric adenocarcinoma is efficient in gastric adenocarcinoma therapy.PI3Kp85αcan be a candidate gene for gene therapy of gastric adenocarcinoma,which will have potential application prospects in clinics.
IA型PI3K/Akt通路与肿瘤的发生发展密切相关。该通路调节肿瘤细胞的增殖和存活,其活性增加不仅能导致细胞恶性转化,而且与肿瘤细胞的迁移、黏附、肿瘤血管生成以及细胞外基质的降解等相关。本课题重点研究了胃腺癌中IA型PI3K/Akt通路的异常表达,并应用RNA干涉技术干预该通路中PI3Kp85α的异常激活,观察干预PI3Kp85α后对胃腺癌恶性表型的影响及对裸鼠皮下SGC7901胃腺癌模型的治疗效果。课题分三部分进行:
第一部分应用组织芯片技术结合免疫组化检测IA型PI3K/Akt通路主要成员PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中的表达情况。
第二部分在体外应用RNA干涉技术以oligofectamine介导siRNA靶向PI3Kp85α转染人胃腺癌细胞系SGC7901后,应用realtime PCR检测目的基因PI3Kp85α的敲低情况,并用Western blot和免疫荧光技术检测了RNAi敲低PI3Kp85α的表达后,IA型PI3K/Akt通路主要成员PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2的表达变化:用MTT、annexin V标记、流式细胞术分析了转染前后细胞增殖、凋亡、细胞周期分布的变化:“划痕”法迁移实验和2-D Matrigel法研究细胞运动迁移能力:用Transwell和3-D Mattigel法分析转染前后细胞侵袭能力的变化。
第三部分建立裸鼠胃腺癌皮下动物模型,每4天瘤内注射oligofectamine和siRNA的混合物一次并测量肿瘤体积,动态观察肿瘤生长情况,治疗32天后切除肿瘤。用免疫组化法检测RNA干涉技术敲低SGC7901裸鼠胃腺癌PI3Kp85α的表达后该通路主要成员的表达变化,并应用TUNEL法检测细胞凋亡,对体外实验的结果进一步验证。
结果:
第一部分组织芯片结合免疫组化技术显示IA型PI3K/Akt通路中PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中均呈高表达,其表达水平随肿瘤的演进程度及恶性级别增高而增强。
第二部分Realtime PCR结果提示,转染PI3Kp85αsiRNA后可明显敲低染PI3Kp85αmRNA的表达。Western blot和免疫荧光检测发现除PI3Kp85α的蛋白明显敲低外,IA型PI3K/Akt通路中pAkt、Ki67、Bcl-2、MMP-2的表达水平均明显降低。MTT结果显示PI3Kp85αsiRNA组与转染nonsense siRNA组及对照组相比细胞增殖率明显受抑(P＜0.01);Annexin V检测显示PI3Kp85αsiRNA组细胞凋亡率明显升高,进一步应用流式细胞术检测细胞周期分布发现S期细胞比例降低,细胞阻滞于G0/G1期;“划痕”法迁移实验和2-D Matrigel法显示PI3Kp85αsiRNA组较nonsense siRNA组和对照组的细胞的运动迁移能力降低(P＜0.01);Transwell和3-D Matrigel法显示其侵袭能力减弱(P＜0.01)。
第三部分成功建立SGC7901胃腺癌细胞系裸鼠皮下肿瘤后,每4天测量一次肿瘤体积,并在肿瘤局部多点注射siRNA和oligofectamine混合物,发现和对照组相比,PI3Kp85αsiRNA治疗组肿瘤生长缓慢,从治疗后的第20天起,肿瘤体积出现明显差别,这种差别在观察末期(32天)最显著。应用免疫组化法检测肿瘤标本,发现PI3Kp85α的蛋白明显敲低的同时,IA型PI3K/Akt通路中pAkt、Ki67、Bcl-2、MMP-2的表达水平均明显降低。应用TUNEL法检测原位细胞凋亡发现PI3Kp85α治疗组凋亡细胞明显增加。体内和体外实验的结果是一致的。
结论:
1.免疫组化显示PI3Kp85α、pAkt、Ki67、Bcl-2、MMP-2在胃腺癌中都有过表达,表明IA型PI3K/Akt信号通路在胃腺癌中是激活的,从而导致细胞增殖和侵袭。其中各种蛋白的表达相互关联也验证了这一点。
2.应用RNA干涉技术以Oligofectamine介导siRNA靶向PI3Kp85α可有效敲低这个基因在人胃腺癌SGC7901细胞内的表达,同时抑制IA型PI3K/Akt通路的活性,从而抑制了细胞的增殖和侵袭,并诱发细胞凋亡。
3.裸鼠皮下胃腺癌模型应用oligofectamine介导的PI3Kp85αsiRNA治疗,同样可有效敲低PI3Kp85α的表达,抑制IA型PI3K/Akt通路的活性,从而抑制肿瘤的生长,诱导细胞凋亡,与体外试验结果一致。
4.通过RNA干涉技术敲低PI3Kp85α的表达对胃腺癌的治疗是有效的。PI3Kp85α可成为胃腺癌基因治疗的候选基因,具有潜在的临床应用前景。
Gastric carcinoma is the most common and incurable digestive systerm tumor,which mortality is the highest.Studying its pathogenesis and trying to find more effective therapeutic modalities are the major research projects in medical field. Gastric carcinoma,just like tumors in other sites of the body,are involved in the processes of uncontrolled cell proliferation,cell dedifferentiation and dysregulation of cell apoptosis.With the development of molecular biology and molecular genetics,it has been found that some of the important oncogenes and tumor suppressor genes are related to the genesis of gastric carcinoma.However,up to date,it is still not clear which are the primary initiating molecular events and which are the secondary promoting molecular events.Therefore,seeking more genes associated with the genesis of gastric carcinoma,a comprehensive understanding of the molecular pathology of gastric carcinoma,and optimizing treatment strategies and developing novel therapeutic approaches for gastric carcinoma on this basis,have become an important research project for carcinoma.
IA Type PI3K/Akt signaling pathway which regulates the proliferation and survival of tumor cells is closely related to the genesis and development of gastric adenocarcinoma.Its activity abnormality not only can induce cells malignant transfor -mation,but also influence the migration and adhesion of tumor cells,vascularization and degradation of extracellular matrix.The present study focused on the abnormal IA Type PI3K/Akt signaling pathway activity in gastric adenocarcinoma and siRNA targeting PI3Kp85αwas used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells and therapeutic efficacy of nude mice subcutaneous SGC7901 gastric adenocarcinoma model.The study was divided into 3 parts.
In the first part,Using tissue microarray techniques and immunohistochemistry assay to survey the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway during gastric adenocarcinoma malignant progression.
In the second part,RNAi technology was used to observe its inhibitory effect on the growth of human gastric adenocarcinoma SGC7901 cells,siRNA targeting PI3Kp85αwas transfected into SGC7901 cells mediated by oligofectamine. PI3Kp85αmRNA expression were detected by realtime PCR after transfection.The expression of PI3Kp85αand other main downstream members including pAkt,Ki67, bcl-2,MMP-2 in IA type PI3K/Akt pathway was also studied by Western blotting and immunofluorescence staining after transfection.The phenotypic change of SGC7901 cells including proliferation,apoptosis and cell cycle after RNAi trasfection was studied by MTT assay,annexin V staining,flow cytometry;their motion and migra -tion ability was studied by scratch assay and 2-D Matrigel assay;the invasion ability was detected by Transwell analysis and 3-D Matrigel assay.
In the third part,subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice.Every 4 days the mixture of oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured.On 32~(nd)day after the first injection,tumors were resected.The expression of PI3Kp85α,pAkt,Ki67,Bcl-2, MMP-2 in IA type PI3K/Akt signaling pathway were studied by immunohistochemis -try.Apoptosis in tumors were detected by TUNEL method.
Results:
The first part:The expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 in IA type PI3K/Akt signaling pathway were overexpressed during gastric adenocarcinoma malignant progression.
The second part:SGC7901 cells were transfected with siRNA targeting PI3Kp85αmediated by oligofectamine in vitro.SGC7901 mRNA expression were obviously knocked down after transfection with siRNA.SGC7901 ceils transfected with siRNA targeting PI3Kp85αshowed lowering proliferation activity by MTT, while the expression of PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2 were downregulated by Western blot analysis and immunohistochemical staining.The transfected cells had higher apotosis rate by annexin V staining and most cells arresting in the Go/G1phase by flow cytometry.The migration and invasive ability was attenuated by scratch assay,2-D Matrigel assay,Transwell analysis and 3-D Matrigel assay.
The third part:Subcutaneous SGC7901 gastric adenocarcinoma model was established in nude mice for in vivo study.As compared with the control group,the tumors in mice treated with siRNA targeting PI3Kp85αgrew slowly and the different ce of tumor volumes became significant since the 20th day after the first time of siRNA therapy until the 32~(nd)day tumors were resected.PI3Kp85α,pAkt,Ki67, Bcl-2,MMP-2's expression were downregulated by immunohistochemistry. Meanwhile,cell apoptosis was increased by TUNEL method.
Conclusion:
1.PI3Kp85α,pAkt,Ki67,Bcl-2,MMP-2's overexpression can lead to IA type PI3K/Akt signaling pathway activation in gastric adenocarcinoma and result in cell proliferation and invasion.Meanwhile,the several proteins in this signaling pathway links to each other and maybe they are modulated with each other.
2.Using RNAi technology,siRNA targeting PI3Kp85αmediated by oligofectamine efficiently knocks down the expression of PI3Kp85αin human gastric adenocarcinoma SGC7901 cells,inhibits activation of IA type PI3K/Akt signaling activity,results in decrease of cell proliferation activity and attenuateion of invasive and migration ability,and induces apoptosis.
3.The established subcutaneous SGC7901 gastric adenocarcinoma models in nude mice are treated with siRNA targeting PI3Kp85α.The tumor growth is inhibited and cell apoptosis is induced.The findings of in vivo study is in accordance with those in vitro study
4.Using RNAi technology to knock down the expression of PI3Kp85αin gastric adenocarcinoma is efficient in gastric adenocarcinoma therapy.PI3Kp85αcan be a candidate gene for gene therapy of gastric adenocarcinoma,which will have potential application prospects in clinics.
引文
[1]Scherr M,Morgan MA,Eder M.Gene silencing mediated by small interfering RNAs in mammalian cells[J].Curr Med Chem,2003,10(3):245-256.
[2]Caplen NJ.RNAi as a gene therapy approach[J].Expert Opin Biol Ther,2003,3(4):575-586.
[3]Luo J,Cantley LC.The negative regulation of phosphoinositide 3-kinase signaling by p85 and it's implication in cancer[J].Cell Cycle,2005,4(10):1309-1312.
[4]Geering B,Cutillas PR,Nock G,et al.Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers[J].Proc Natl Acad Sci USA,2007,104(19):7809-7814.
[5]于士柱,浦佩玉,江德华,等.胃腺癌基因表达水平与其细胞增殖和凋亡关系的研究[J].中华病理学杂志,2000,29(1):12-15.
[6]Shukla S,Maclennan GT,Hartman DJ,et al.Activation of PI3K-Akt signaling pathway promotes prostate cancer cell invasion[J].Int J Cancer,2007,121(7):1424-1432.
[7]Philp AJ,Campbell IG,Leet C,et al.The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors[J].Cancer Res,2001,61(20):7426-7429.
[8]Kobayashi I,Semba S,Matsuda Y,et al.Significance of Akt phosphorylation on tumor growth and vascular endothelial growth factor expression in human gastric carcinoma[J].Pathobiology,2006,73(1):8-17.
[9]Perez-Tenorio G,Stal O.Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients[J].Br J Cancer,2002,86(4):540-545.
[10]Murakami D,Tsujitani S,Osaki T,et al.Expression of phosphorylated Akt(pAkt)in gastric carcinoma predicts prognosis and efficacy of chemotherapy[J].Gastric Cancer,2007,10(1):45-51.
[11]Kumar A,Carrera AC.New functions for PI3K in the control of cell division[J].Cell Cycle,2007,6(14):1696-1698.
[12] Witte F,Honig A,Mirecka J,et al. Cystosarcoma phyllodes of the breast: prognostic significance of proliferation and apoptosis associated genes[J]. Anticancer Res,1999,19(4B):3355-3359.
[13] Shpitz B, Bomstein Y, Sternberg A,et al. Immunoreactivity of p53, Ki-67, and c-erbB-2 in phyllodes tumors of the breast in correlation with clinical and morphologic features[J]. J Surg Oncol,2002,79(2):86-92.
[14] Monig SP,Baldus SE,Hennecken JK,et al. Expression of MMP-2 is associated with progression and lymph node metastasis of gastric carcinoma[J]. Histopathology, 2001,39(6):597-602.
[15] Feng G,Tan Y. Expression and significance of MMP2 and type IV collagen in gastric cancer[J]. Zhonghua Wai Ke Za Zhi,2000,38(10):775-777.
[16] Yang CQ, Wang JY, Fang GT, et al. A comparison between intravenous and peritoneal route on liver targeted uptake and expression of plasmid delivered by Glyco-poly-l-lysine[J]. World J Gastroenterol,2000,6(4):508-512.
[17] Xu J,Xu Z,Jiang Y,et al. Cryptorchidism induces mouse testicular germ cell apoptosis and changes in bcl-2 and bax protein expression[J]. J Environ Pathol Toxicol Oncol,2000,19(12):25-33.
[18] Kasagi N,Gomyo Y,Shirai H,et al. Apoptotic cell death in human gastric carcinoma: analysis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling[J]. Jpn J Cancer Res,1994,85(9):939-945.
[19] Saegusa M,Takano Y,Wakabayashi t,et al. Apoptosis in gastric carcinomas and its association with cell proliferation and differentiation[J]. Jpn J Cancer Res, 1995,86(8): 743-748.
[20] Boado RJ. RNA interference and nonviral targeted gene therapy of experimental brain cancer[J]. NeuroRx,2005,2(1):139-150.
[21] Bernstein E,Caudy AA,Hammond SM,et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature,2001,409(6818):363-366.
[22] Hannon GJ.RNA interference[J].Nature,2002,418(6894):244-251.
[23] Sijen T,Fleenor J,Simmer F,et al. On the role of RNA amplification in dsRNA-triggered gene silencing [J]. Cell,2001,107(4):465-476.
[24] Ramaswamy G,Slack FJ. siRNA:A guide for RNA silencing [J]. Chem Biol, 2002,9(10):1053-1055.
[25] Caplen NJ,Parrish Simani F,et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems[J]. Proc Natl Acad Sci USA,2001,98(17):9742-9747.
[26] Gartel AL, Kandel ES. RNA interference in cancer[J]. Biomol Eng,2006,23(1): 17-34.
[27] Zheng ZH,Sun XJ,Zhou HT,et al. Analysis of metastasis suppressing function of E-cadherin in gastric cancer cells by RNAi [J]. World J Gastroenterol,2005,11(13): 2000-2003.
[28] Spankuch-Schmitt B, Bereiter-Hahn J, Kaufmann M,et al. Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells [J]. J Natl Cancer Inst,2002,94(24):1863-1877.
[29] Brummelkamp TR,Bernards R,Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell,2002,2(3):243-247.
[30] Lin SL,Chuong CM,Ying SY. A Novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells [J]. Biochem Biophys Res Commun,2001,281(3):639-644.
[31] Wannenes F,Ciafre SA,Niola F,et al. Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo [J]. Cancer Gene Ther,2005,12(12):926-934.
[32] Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase[J]. Nucleic Acids Res,2002,30(10):46-50.
[33] Sohail M, Doran G, Riedemann J, et al. A simple and cost-effective method for producing small interfering RNAs with high efficacy [J]. Nucleic Acids Res,2003, 31(7):38-44.
[34] Kim DH, Longo M, Han Y, et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase[J], Nat Biotechnol,2004,22(3):321-325.
[35] Braasch DA, Jensen S, Liu Y, et al. RNA interference in mammalian cells by chemically-modified RNA[J]. Biochemistry,2003,42(26):7967-7975.
[36]Chiu YL, Rana TM. siRNA function in RNAi: a chemical modification analysis. RNA, 2003,9(9): 1034-1048.
[37] 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(23):6877-6888.
[38] Holen T, Amarzguioui M, Wiiger MT, et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor[J]. Nucleic Acids Res, 2002,30(8): 1757-1766.
[39] Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing[J]. Antisense Nucleic Acid Drug Dev, 2003,13(2): 83-105.
[40] Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells [J]. Proc Natl Acad Sci USA, 2002,99(9):6047-6052.
[41] Lingor P, Michel U, Scholl U, et al. Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons[J]. Biochem Biophys Res Commun,2004,315(4):1126-1133.
[42] Overhoff M, Wunsche W, Sczakiel G. Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA[J]. Nucleic Acids Res,2004,32(21): 170-177.
[43] Sobol RE, Scanlon KJ. Cancer gene therapy clinical trials [J]. Cancer Gene Ther, 1995,2(1):5-6.
[44] Audouy SA, de Leij LF, Hoekstra D, et al. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[J]. Pharm Res,2002,19(11): 1599-1605.
[45] Hirko A, Tang F, Hughes JA. Cationic lipid vectors for plasmid DNA delivery [J]. Curr Med Chem,2003,10(14): 1185-1193.
[46] Aissaoui A, Oudrhiri N, Petit L, et al. Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example[J]. Curr Drug Targets,2002, 3(1):1-16.
[47] Pedroso de Lima MC, Simoes S, Pires P, et al. Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications[J]. Adv Drug Deliv Rev, 2001,47(23):277-294.
[48] Duzgunes N, De Ilarduya CT, Simoes S, et al. Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides[J]. Curr Med Chem,2003,10(14):1213-1220.
[49] Beale G, Hollins AJ, Benboubetra M, et al. Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system[J], J Drug Target,2003,11(7):449-456.
[50] 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.
[51] Philipp V,Carsten B,Bernd N,et al.Assigning functional domains within the p110 regulatory subunit of phosphoinositide 3-kinase[J].J Biol Chem,2005,280(6): 5121- 5127.
[52]] Ringel MD,Hayre N,Saito J,et al. Overexpression and overactivation of Akt in thyroid carcinoma [J]. Cancer Res, 2001,61(16):6105-6111.
[53] Yuan ZQ,Sun M,Feldman RI,et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer [J]. Oncogene,2000,19(19):2324-2330.
[54] Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation [J]. J Biol Chem,2005,280(11):10781-10789.
[55] Loberg RD, Vesely E, Brosius FC,et al.Enhanced glycogen synthase kinase-3 beta activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism[J].J Biol Chem, 2002,277(44): 41667-41673.
[56] Battling B,Tostlebe H,Darmer D,et al. Shear stress-dependent expression of apoptosis-regulating genes in endothelial cells [J]. Biochem Biophys Res Commun, 2000,278(3):740-746.
[57] Li N,Banin S,Ouyang H,et al. ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks [J]. J Biol Chem,2001,276(12): 8898-903.
[58] Gibson EM,Henson ES,Haney N,et al. Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release [J]. Cancer Res, 2002,62 (2):488-496.
[59] Coffey JC,Wang JH,Smith MJ,et al. Phosphoinositide 3-kinase accelerates postoperative tumor growth by inhibiting apoptosis and enhancing resistance to chemotherapy-induced apoptosis. Novel role for an old enemy [J]. J Biol Chem,2005, 280(22):20968-20977.
[60] Mayo LD,Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus [J]. Proc Natl Acad Sci USA, 2001,98(20): 11598-11603.
[61] Lee D,Yang D,Kang C,et al. Serum insulin-like growth factors (IGFs) and IGF binding protein (IGFBP)-3 in patients with gastric cancer: IGFBP-3 protease activity induced by surgery [J]. J Korean Med Sci,1997 ,12(1):32-39.
[62] Zhang D,Brodt P. Type 1 insulin-like growth factor regulates MT1-MMP synthesis and tumor invasion via PI 3-kinase/Akt signaling [J]. Oncogene,2003,22(7): 974-982.
[63] Kim D,Kim S,Koh H,et al. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production [J]. FASEB J,2001,15(11): 1953-1962.
[64] Sheehan KM,Sheahan K,O'Donoghue DP,et al. The relationship between cyclooxygenase-2 expression and colorectal cancer [J]. JAMA,1999,282(13):1254- 1257.
[65] Hill MM,Hemmings BA. Inhibition of protein kinase B/Akt. implications for cancer therapy [J]. Pharmacol Ther,2002,93(23):243-251.
[66] Kim CS,Vasko VV,Kato Y,et al. AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma [J]. Endocrinology,2005,146(10):4456-4463.
[67] Qian Y,Corum L,Meng Q,et al. PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration [J]. Am J Physiol Cell Physiol,2004,286(1): 153-163.
[68] Pantelouris EM. Absence of thymus in a mouse mutant[J]. Nature, 1968, 217 (5126):370-371.
[69] Raygard J,Povlsen CO. Heterotransplantation of a human malignant tumour to "Nude" mice[J]. Acta Pathol Microbiol Scand,1969,77(4):758-760.
[1] Lee Y, Kim M, Han J, et al. MiRNA genes are transcribed by RNA polymerase II[J]. EMBO J,2004 ,23(20):4051-4060.
[2] Gregory RI, Shiekhattar R.MiRNA biogenesis and Cancer[J]. Cancer Res,2005,65(9):3509-3512.
[3] Kent OA,Mendell JT. A small piece in the cancer puzzle:miRNA as tumor suppressors and oncogens[J]. Oncogene,2006,25(46):6188-6196.
[4] Du T, Zamore PD.MicroPriner:the biogenesis and function of miRNA[J]. Development,2005,132(21):4645-4652.
[5] Tomari Y, Zamore PD. Perspective: Machines for RNAi[J]. Genes Dev, 2005, 19(5):517-529.
[6] Bartel D P. MiRNA: Genomics, biogenesis, mechanism, and function[J]. Cell,2004, 116(2):281-297.
[7] Wu L, Fan J, Belasco JG. MiRNA direct rapid deadenylation of Mrna[J]. Proc Natl Acad Sci USA, 2006, 103(11):4034-4039.
[8] Calin GA,Sevignani C, Dumitru CD, et al. Human miRNA genes are frequently located at fragile sites and genomic regions involved in cancers [J]. Proc Natl Acad Sci U S A,2004, 101(9):2999-3004.
[9] Volinia S, Calin GA, Liu CG, et al. A miRNA expression signature of human solid tumors defines cancer gene targets[J]. Proc Natl Acad Sci USA, 2006 , 103(7):2257-2261.
[10] Costinean S, Zanesi N, Pekarsky Y, et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice[J]. Proc Natl Acad Sci U S A,2006 103(18):7024-7029.
[11] Martin MM, Lee EJ, Buckenberger JA, et al. MiRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts[J]. J Biol Chem, 2006, 281(27): 18277-18284.
[12] Ota A, Tagawa H, Karnan S,et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma [J]. Cancer Res,2004, 64(9):3087-3095.
[13] Hayashita Y, Osada H, Tatematsu Y,et al. A polycistronic miRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res, 2005, 65(21):9628-9632.
[14] Chan JA, Krichevsky AM, Kosik KS. MiRNA-21 is an antiapoptotic factor in human glioblastoma cells[J]. Cancer Res, 2005, 65(14):6029-6033.
[15] Voorhoeve PM, Le Sage C, Schrier M,et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors [J]. Adv Exp Med Biol,2007,604:17-29.
[16] Calin GA,Noch E,Yendamuri S,et al. Human miRNA genes are frequently located at fragile sites and genomic regions involved in cancers [J]. Proc Natl Acad Sci USA, 2004 ,101(9):2999-3004.
[17] Zhang B, Pan X, Cobb GP, et al. miRNA as oncogenes and tumor suppressors[J]. Dev Biol, 2007, 302(1): 1-13.
[18] Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 miRNA in human lung cancers in association with shortened postoperative survival [J]. Cancer Res, 2004, 64(11):3753-3756.
[19] Yanaihara N, Caplen N, Bowman E, et al. Unique miRNA molecular profilesin lung cancer diagnosis and prognosis[J]. Cancer Cell, 2006, 9(3): 189-198.
[20] Michael MZ, O' Connor SM, van Hoist Pellekaan NG, et al. Reduced accumulation of specific miRNA in colorectal neoplasia[J]. Mol Cancer Res, 2003,1(12):882-891.
[21] Dalmay T, Edwards DR. MiRNA and the hallmarks of cancer[J]. Oncogene, 2006, 25(46):6170-6175.
[2]Caplen NJ.RNAi as a gene therapy approach[J].Expert Opin Biol Ther,2003,3(4):575-586.
[3]Luo J,Cantley LC.The negative regulation of phosphoinositide 3-kinase signaling by p85 and it's implication in cancer[J].Cell Cycle,2005,4(10):1309-1312.
[4]Geering B,Cutillas PR,Nock G,et al.Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers[J].Proc Natl Acad Sci USA,2007,104(19):7809-7814.
[5]于士柱,浦佩玉,江德华,等.胃腺癌基因表达水平与其细胞增殖和凋亡关系的研究[J].中华病理学杂志,2000,29(1):12-15.
[6]Shukla S,Maclennan GT,Hartman DJ,et al.Activation of PI3K-Akt signaling pathway promotes prostate cancer cell invasion[J].Int J Cancer,2007,121(7):1424-1432.
[7]Philp AJ,Campbell IG,Leet C,et al.The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors[J].Cancer Res,2001,61(20):7426-7429.
[8]Kobayashi I,Semba S,Matsuda Y,et al.Significance of Akt phosphorylation on tumor growth and vascular endothelial growth factor expression in human gastric carcinoma[J].Pathobiology,2006,73(1):8-17.
[9]Perez-Tenorio G,Stal O.Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients[J].Br J Cancer,2002,86(4):540-545.
[10]Murakami D,Tsujitani S,Osaki T,et al.Expression of phosphorylated Akt(pAkt)in gastric carcinoma predicts prognosis and efficacy of chemotherapy[J].Gastric Cancer,2007,10(1):45-51.
[11]Kumar A,Carrera AC.New functions for PI3K in the control of cell division[J].Cell Cycle,2007,6(14):1696-1698.
[12] Witte F,Honig A,Mirecka J,et al. Cystosarcoma phyllodes of the breast: prognostic significance of proliferation and apoptosis associated genes[J]. Anticancer Res,1999,19(4B):3355-3359.
[13] Shpitz B, Bomstein Y, Sternberg A,et al. Immunoreactivity of p53, Ki-67, and c-erbB-2 in phyllodes tumors of the breast in correlation with clinical and morphologic features[J]. J Surg Oncol,2002,79(2):86-92.
[14] Monig SP,Baldus SE,Hennecken JK,et al. Expression of MMP-2 is associated with progression and lymph node metastasis of gastric carcinoma[J]. Histopathology, 2001,39(6):597-602.
[15] Feng G,Tan Y. Expression and significance of MMP2 and type IV collagen in gastric cancer[J]. Zhonghua Wai Ke Za Zhi,2000,38(10):775-777.
[16] Yang CQ, Wang JY, Fang GT, et al. A comparison between intravenous and peritoneal route on liver targeted uptake and expression of plasmid delivered by Glyco-poly-l-lysine[J]. World J Gastroenterol,2000,6(4):508-512.
[17] Xu J,Xu Z,Jiang Y,et al. Cryptorchidism induces mouse testicular germ cell apoptosis and changes in bcl-2 and bax protein expression[J]. J Environ Pathol Toxicol Oncol,2000,19(12):25-33.
[18] Kasagi N,Gomyo Y,Shirai H,et al. Apoptotic cell death in human gastric carcinoma: analysis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling[J]. Jpn J Cancer Res,1994,85(9):939-945.
[19] Saegusa M,Takano Y,Wakabayashi t,et al. Apoptosis in gastric carcinomas and its association with cell proliferation and differentiation[J]. Jpn J Cancer Res, 1995,86(8): 743-748.
[20] Boado RJ. RNA interference and nonviral targeted gene therapy of experimental brain cancer[J]. NeuroRx,2005,2(1):139-150.
[21] Bernstein E,Caudy AA,Hammond SM,et al. Role for a bidentate ribonuclease in the initiation step of RNA interference [J]. Nature,2001,409(6818):363-366.
[22] Hannon GJ.RNA interference[J].Nature,2002,418(6894):244-251.
[23] Sijen T,Fleenor J,Simmer F,et al. On the role of RNA amplification in dsRNA-triggered gene silencing [J]. Cell,2001,107(4):465-476.
[24] Ramaswamy G,Slack FJ. siRNA:A guide for RNA silencing [J]. Chem Biol, 2002,9(10):1053-1055.
[25] Caplen NJ,Parrish Simani F,et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems[J]. Proc Natl Acad Sci USA,2001,98(17):9742-9747.
[26] Gartel AL, Kandel ES. RNA interference in cancer[J]. Biomol Eng,2006,23(1): 17-34.
[27] Zheng ZH,Sun XJ,Zhou HT,et al. Analysis of metastasis suppressing function of E-cadherin in gastric cancer cells by RNAi [J]. World J Gastroenterol,2005,11(13): 2000-2003.
[28] Spankuch-Schmitt B, Bereiter-Hahn J, Kaufmann M,et al. Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells [J]. J Natl Cancer Inst,2002,94(24):1863-1877.
[29] Brummelkamp TR,Bernards R,Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell,2002,2(3):243-247.
[30] Lin SL,Chuong CM,Ying SY. A Novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells [J]. Biochem Biophys Res Commun,2001,281(3):639-644.
[31] Wannenes F,Ciafre SA,Niola F,et al. Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo [J]. Cancer Gene Ther,2005,12(12):926-934.
[32] Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase[J]. Nucleic Acids Res,2002,30(10):46-50.
[33] Sohail M, Doran G, Riedemann J, et al. A simple and cost-effective method for producing small interfering RNAs with high efficacy [J]. Nucleic Acids Res,2003, 31(7):38-44.
[34] Kim DH, Longo M, Han Y, et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase[J], Nat Biotechnol,2004,22(3):321-325.
[35] Braasch DA, Jensen S, Liu Y, et al. RNA interference in mammalian cells by chemically-modified RNA[J]. Biochemistry,2003,42(26):7967-7975.
[36]Chiu YL, Rana TM. siRNA function in RNAi: a chemical modification analysis. RNA, 2003,9(9): 1034-1048.
[37] 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(23):6877-6888.
[38] Holen T, Amarzguioui M, Wiiger MT, et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor[J]. Nucleic Acids Res, 2002,30(8): 1757-1766.
[39] Harborth J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing[J]. Antisense Nucleic Acid Drug Dev, 2003,13(2): 83-105.
[40] Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells [J]. Proc Natl Acad Sci USA, 2002,99(9):6047-6052.
[41] Lingor P, Michel U, Scholl U, et al. Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons[J]. Biochem Biophys Res Commun,2004,315(4):1126-1133.
[42] Overhoff M, Wunsche W, Sczakiel G. Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA[J]. Nucleic Acids Res,2004,32(21): 170-177.
[43] Sobol RE, Scanlon KJ. Cancer gene therapy clinical trials [J]. Cancer Gene Ther, 1995,2(1):5-6.
[44] Audouy SA, de Leij LF, Hoekstra D, et al. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy[J]. Pharm Res,2002,19(11): 1599-1605.
[45] Hirko A, Tang F, Hughes JA. Cationic lipid vectors for plasmid DNA delivery [J]. Curr Med Chem,2003,10(14): 1185-1193.
[46] Aissaoui A, Oudrhiri N, Petit L, et al. Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example[J]. Curr Drug Targets,2002, 3(1):1-16.
[47] Pedroso de Lima MC, Simoes S, Pires P, et al. Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications[J]. Adv Drug Deliv Rev, 2001,47(23):277-294.
[48] Duzgunes N, De Ilarduya CT, Simoes S, et al. Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides[J]. Curr Med Chem,2003,10(14):1213-1220.
[49] Beale G, Hollins AJ, Benboubetra M, et al. Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system[J], J Drug Target,2003,11(7):449-456.
[50] 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.
[51] Philipp V,Carsten B,Bernd N,et al.Assigning functional domains within the p110 regulatory subunit of phosphoinositide 3-kinase[J].J Biol Chem,2005,280(6): 5121- 5127.
[52]] Ringel MD,Hayre N,Saito J,et al. Overexpression and overactivation of Akt in thyroid carcinoma [J]. Cancer Res, 2001,61(16):6105-6111.
[53] Yuan ZQ,Sun M,Feldman RI,et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer [J]. Oncogene,2000,19(19):2324-2330.
[54] Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation [J]. J Biol Chem,2005,280(11):10781-10789.
[55] Loberg RD, Vesely E, Brosius FC,et al.Enhanced glycogen synthase kinase-3 beta activity mediates hypoxia-induced apoptosis of vascular smooth muscle cells and is prevented by glucose transport and metabolism[J].J Biol Chem, 2002,277(44): 41667-41673.
[56] Battling B,Tostlebe H,Darmer D,et al. Shear stress-dependent expression of apoptosis-regulating genes in endothelial cells [J]. Biochem Biophys Res Commun, 2000,278(3):740-746.
[57] Li N,Banin S,Ouyang H,et al. ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks [J]. J Biol Chem,2001,276(12): 8898-903.
[58] Gibson EM,Henson ES,Haney N,et al. Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release [J]. Cancer Res, 2002,62 (2):488-496.
[59] Coffey JC,Wang JH,Smith MJ,et al. Phosphoinositide 3-kinase accelerates postoperative tumor growth by inhibiting apoptosis and enhancing resistance to chemotherapy-induced apoptosis. Novel role for an old enemy [J]. J Biol Chem,2005, 280(22):20968-20977.
[60] Mayo LD,Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus [J]. Proc Natl Acad Sci USA, 2001,98(20): 11598-11603.
[61] Lee D,Yang D,Kang C,et al. Serum insulin-like growth factors (IGFs) and IGF binding protein (IGFBP)-3 in patients with gastric cancer: IGFBP-3 protease activity induced by surgery [J]. J Korean Med Sci,1997 ,12(1):32-39.
[62] Zhang D,Brodt P. Type 1 insulin-like growth factor regulates MT1-MMP synthesis and tumor invasion via PI 3-kinase/Akt signaling [J]. Oncogene,2003,22(7): 974-982.
[63] Kim D,Kim S,Koh H,et al. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production [J]. FASEB J,2001,15(11): 1953-1962.
[64] Sheehan KM,Sheahan K,O'Donoghue DP,et al. The relationship between cyclooxygenase-2 expression and colorectal cancer [J]. JAMA,1999,282(13):1254- 1257.
[65] Hill MM,Hemmings BA. Inhibition of protein kinase B/Akt. implications for cancer therapy [J]. Pharmacol Ther,2002,93(23):243-251.
[66] Kim CS,Vasko VV,Kato Y,et al. AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma [J]. Endocrinology,2005,146(10):4456-4463.
[67] Qian Y,Corum L,Meng Q,et al. PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration [J]. Am J Physiol Cell Physiol,2004,286(1): 153-163.
[68] Pantelouris EM. Absence of thymus in a mouse mutant[J]. Nature, 1968, 217 (5126):370-371.
[69] Raygard J,Povlsen CO. Heterotransplantation of a human malignant tumour to "Nude" mice[J]. Acta Pathol Microbiol Scand,1969,77(4):758-760.
[1] Lee Y, Kim M, Han J, et al. MiRNA genes are transcribed by RNA polymerase II[J]. EMBO J,2004 ,23(20):4051-4060.
[2] Gregory RI, Shiekhattar R.MiRNA biogenesis and Cancer[J]. Cancer Res,2005,65(9):3509-3512.
[3] Kent OA,Mendell JT. A small piece in the cancer puzzle:miRNA as tumor suppressors and oncogens[J]. Oncogene,2006,25(46):6188-6196.
[4] Du T, Zamore PD.MicroPriner:the biogenesis and function of miRNA[J]. Development,2005,132(21):4645-4652.
[5] Tomari Y, Zamore PD. Perspective: Machines for RNAi[J]. Genes Dev, 2005, 19(5):517-529.
[6] Bartel D P. MiRNA: Genomics, biogenesis, mechanism, and function[J]. Cell,2004, 116(2):281-297.
[7] Wu L, Fan J, Belasco JG. MiRNA direct rapid deadenylation of Mrna[J]. Proc Natl Acad Sci USA, 2006, 103(11):4034-4039.
[8] Calin GA,Sevignani C, Dumitru CD, et al. Human miRNA genes are frequently located at fragile sites and genomic regions involved in cancers [J]. Proc Natl Acad Sci U S A,2004, 101(9):2999-3004.
[9] Volinia S, Calin GA, Liu CG, et al. A miRNA expression signature of human solid tumors defines cancer gene targets[J]. Proc Natl Acad Sci USA, 2006 , 103(7):2257-2261.
[10] Costinean S, Zanesi N, Pekarsky Y, et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice[J]. Proc Natl Acad Sci U S A,2006 103(18):7024-7029.
[11] Martin MM, Lee EJ, Buckenberger JA, et al. MiRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts[J]. J Biol Chem, 2006, 281(27): 18277-18284.
[12] Ota A, Tagawa H, Karnan S,et al. Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma [J]. Cancer Res,2004, 64(9):3087-3095.
[13] Hayashita Y, Osada H, Tatematsu Y,et al. A polycistronic miRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation[J]. Cancer Res, 2005, 65(21):9628-9632.
[14] Chan JA, Krichevsky AM, Kosik KS. MiRNA-21 is an antiapoptotic factor in human glioblastoma cells[J]. Cancer Res, 2005, 65(14):6029-6033.
[15] Voorhoeve PM, Le Sage C, Schrier M,et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors [J]. Adv Exp Med Biol,2007,604:17-29.
[16] Calin GA,Noch E,Yendamuri S,et al. Human miRNA genes are frequently located at fragile sites and genomic regions involved in cancers [J]. Proc Natl Acad Sci USA, 2004 ,101(9):2999-3004.
[17] Zhang B, Pan X, Cobb GP, et al. miRNA as oncogenes and tumor suppressors[J]. Dev Biol, 2007, 302(1): 1-13.
[18] Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 miRNA in human lung cancers in association with shortened postoperative survival [J]. Cancer Res, 2004, 64(11):3753-3756.
[19] Yanaihara N, Caplen N, Bowman E, et al. Unique miRNA molecular profilesin lung cancer diagnosis and prognosis[J]. Cancer Cell, 2006, 9(3): 189-198.
[20] Michael MZ, O' Connor SM, van Hoist Pellekaan NG, et al. Reduced accumulation of specific miRNA in colorectal neoplasia[J]. Mol Cancer Res, 2003,1(12):882-891.
[21] Dalmay T, Edwards DR. MiRNA and the hallmarks of cancer[J]. Oncogene, 2006, 25(46):6170-6175.