AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines wi...AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium(MTT) assay.The expression changes of Smad2,Smad3,Smad4,Smad7,TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line(MIHA),a TGF-β-sensitive hepatoma cell line(Hep3B) and two TGF-β-insensitive hepatoma cell lines(HepG2 and Bel7404) were examined.SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined.Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines(Bel7404 and HepG2).MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis,respectively.The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis,and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.RESULTS:TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line,Hep3B,but not in the resistant cell lines.The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1,whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines,which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1.Our data further suggested that stathmin was a direct target of TIEG1,as stathmin was signif icantly downregulated by TIEG1 overexpression,and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.CONCLUSION:Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.展开更多
Objective To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tis- sues and to validate its bona fide targets in human cholangiocarcinoma cells. Methods The expression profile of microRNA...Objective To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tis- sues and to validate its bona fide targets in human cholangiocarcinoma cells. Methods The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholan- giocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respec- tively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarci- noma tissues by using real-time PCR, locked nucleic acid in situ hybridization （LNA-ISH）, and immunohis- tochemistry analysis. Results Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bileduct tissues （P〈0.05）. The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholan- giocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN （P〈0.01） and PDCD4 （P〈0.05） and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue. Conclusion microRNA-21 expression is up PDCD4 are direct effectors of microRNA-21. regulated in human cholangiocarcinoma and PTEN,展开更多
AIM： To investigate the molecular mechanism and functional consequences of heme oxygenase-1 （HO-1） activation by lansoprazole in endothelial cells and macrophages. METHODS： Expression of HO-1 mRNA was analyzed by ...AIM： To investigate the molecular mechanism and functional consequences of heme oxygenase-1 （HO-1） activation by lansoprazole in endothelial cells and macrophages. METHODS： Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species （ROS） formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed usingin vivo bioluminescence imaging. RESULTS： Lansoprazole levels in endothelial cells increased HO-1 mRNA and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH- mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity.CONCLUSION： Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002.展开更多
基金Supported by Hong Kong Research Grant Council,No.467109,467507the Scientif ic Research Fund of Zhejiang Provincial Ed-ucation Department,No.Y200906317+1 种基金the Wenzhou Science and Technology Bureau Program,No.Y20100017Qianjiang Talents Project of Zhejiang Province,No.2011R10058
文摘AIM:To investigate the role of transforming growth factor(TGF)-β-inducible early gene 1(TIEG1) in TGF-β-induced growth inhibition in hepatocellular carcinoma(HCC) cells.METHODS:Human hepatocyte and HCC cell lines with varied susceptibilities to TGF-β1 were tested by methylthiazoletetrazolium(MTT) assay.The expression changes of Smad2,Smad3,Smad4,Smad7,TIEG1 and TIEG2 gene following treatment with TGF-β1 in a TGF-β-sensitive hepatocyte cell line(MIHA),a TGF-β-sensitive hepatoma cell line(Hep3B) and two TGF-β-insensitive hepatoma cell lines(HepG2 and Bel7404) were examined.SiRNA targeting TIEG1 was transfected into Hep3B cells and the sensitivity of cells to TGF-β1 was examined.Overexpression of TIEG1 was induced by lentiviral-mediated transduction in TGF-β1-resistant hepatoma cell lines(Bel7404 and HepG2).MTT assay and 4',6-Diamidino-2-phenylindole staining were used to identify cell viability and apoptosis,respectively.The expression level of stathmin was measured by reverse transcriptase polymerase chain reaction and Western-blotting analysis,and stathmin promoter activity by TIEG1 was monitored by a luciferase reporter gene system.RESULTS:TIEG1 was significantly upregulated by TGF-β1 in the TGF-β1-sensitive HCC cell line,Hep3B,but not in the resistant cell lines.The suppression of TIEG1 by siRNAs decreased the sensitivity of Hep3B cells to TGF-β1,whereas the overexpression of TIEG1 mediated growth inhibition and apoptosis in TGF-β1-resistant HCC cell lines,which resembled those of TGF-β1-sensitive HCC cells treated with TGF-β1.Our data further suggested that stathmin was a direct target of TIEG1,as stathmin was signif icantly downregulated by TIEG1 overexpression,and stathmin promoter activity was inhibited by TIEG1 in a dose-dependent manner.CONCLUSION:Our data suggest that transactivation of TIEG1 conferred growth inhibition of TGF-β-susceptible human HCC cells.
基金Supported by Institute of Basic Medical Sciences(2009PY13 and 2010PYZ18)the National Natural Science Foundation of China(81100608 and 30901342)
文摘Objective To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tis- sues and to validate its bona fide targets in human cholangiocarcinoma cells. Methods The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholan- giocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respec- tively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarci- noma tissues by using real-time PCR, locked nucleic acid in situ hybridization （LNA-ISH）, and immunohis- tochemistry analysis. Results Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bileduct tissues （P〈0.05）. The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholan- giocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN （P〈0.01） and PDCD4 （P〈0.05） and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue. Conclusion microRNA-21 expression is up PDCD4 are direct effectors of microRNA-21. regulated in human cholangiocarcinoma and PTEN,
基金Supported by The German Academic Exchange Program(DAAD,S.S.G.)
文摘AIM： To investigate the molecular mechanism and functional consequences of heme oxygenase-1 （HO-1） activation by lansoprazole in endothelial cells and macrophages. METHODS： Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species （ROS） formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed usingin vivo bioluminescence imaging. RESULTS： Lansoprazole levels in endothelial cells increased HO-1 mRNA and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH- mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity.CONCLUSION： Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002.
