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
人stathmin也称原癌基因蛋白18(oncogene protein 18,Op18),是一种广泛存在于细胞质的蛋白质,其相对分子质量约为19×103,可与微管蛋白结合,参与微管和纺锤体的组装;与细胞的增殖、分化、再生和运动均有关,并具有信号活性调节的功...人stathmin也称原癌基因蛋白18(oncogene protein 18,Op18),是一种广泛存在于细胞质的蛋白质,其相对分子质量约为19×103,可与微管蛋白结合,参与微管和纺锤体的组装;与细胞的增殖、分化、再生和运动均有关,并具有信号活性调节的功能。近年来有研究发现,stathmin在多种肿瘤中高表达,并可通过调节微管的解聚,促进肿瘤细胞的运动及侵袭。Stathmin翻译后修饰状态的改变,可影响与p53蛋白的相互作用,参与肿瘤的发生发展。目前单独或者联合化疗药物使用的抗stathmin效应剂已用于某些肿瘤的治疗。虽然stathmin与肿瘤病因学的内在联系尚不十分清楚,但其作为一个潜在的肿瘤标志物或药物靶点值得进一步研究探讨。展开更多
目的探讨以RNA干扰(RNAinterference,RNAi)技术靶向stathmin和mdr1基因逆转卵巢癌细胞紫杉醇耐药的可行性。方法分别构建靶向stathmin和mdr1基因的质粒:pGU6-GFP-neo-ST-MN1和pGU6-GFP-neo-MDR1;将质粒转染到卵巢癌紫杉醇耐药细胞株SKOV...目的探讨以RNA干扰(RNAinterference,RNAi)技术靶向stathmin和mdr1基因逆转卵巢癌细胞紫杉醇耐药的可行性。方法分别构建靶向stathmin和mdr1基因的质粒:pGU6-GFP-neo-ST-MN1和pGU6-GFP-neo-MDR1;将质粒转染到卵巢癌紫杉醇耐药细胞株SKOV3/TAX。Real-ti me RT-PCR检测stathminm和mdr1的mRNA变化;Western blot检测其蛋白表达变化;荧光显微镜检测细胞凋亡情况;CCK-8法分析细胞对紫杉醇的敏感度。结果 Real-ti me RT-PCR及Western blot显示pGU6-GFP-neo-MDR1质粒对mdr1基因,pGU6-GFP-STMN1-294shRNA质粒对stathmin基因在mR-NA水平和蛋白水平均有明显抑制(P<0.05),荧光显微镜下共转染组细胞凋亡增多,CCK-8示共转染组逆转紫杉醇耐药效果最明显。结论体外RNAi可有效沉默卵巢癌紫杉醇耐药株SKOV3/TAX细胞内stathmin基因和mdr1基因,逆转其紫杉醇耐药。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.
文摘人stathmin也称原癌基因蛋白18(oncogene protein 18,Op18),是一种广泛存在于细胞质的蛋白质,其相对分子质量约为19×103,可与微管蛋白结合,参与微管和纺锤体的组装;与细胞的增殖、分化、再生和运动均有关,并具有信号活性调节的功能。近年来有研究发现,stathmin在多种肿瘤中高表达,并可通过调节微管的解聚,促进肿瘤细胞的运动及侵袭。Stathmin翻译后修饰状态的改变,可影响与p53蛋白的相互作用,参与肿瘤的发生发展。目前单独或者联合化疗药物使用的抗stathmin效应剂已用于某些肿瘤的治疗。虽然stathmin与肿瘤病因学的内在联系尚不十分清楚,但其作为一个潜在的肿瘤标志物或药物靶点值得进一步研究探讨。
文摘目的探讨以RNA干扰(RNAinterference,RNAi)技术靶向stathmin和mdr1基因逆转卵巢癌细胞紫杉醇耐药的可行性。方法分别构建靶向stathmin和mdr1基因的质粒:pGU6-GFP-neo-ST-MN1和pGU6-GFP-neo-MDR1;将质粒转染到卵巢癌紫杉醇耐药细胞株SKOV3/TAX。Real-ti me RT-PCR检测stathminm和mdr1的mRNA变化;Western blot检测其蛋白表达变化;荧光显微镜检测细胞凋亡情况;CCK-8法分析细胞对紫杉醇的敏感度。结果 Real-ti me RT-PCR及Western blot显示pGU6-GFP-neo-MDR1质粒对mdr1基因,pGU6-GFP-STMN1-294shRNA质粒对stathmin基因在mR-NA水平和蛋白水平均有明显抑制(P<0.05),荧光显微镜下共转染组细胞凋亡增多,CCK-8示共转染组逆转紫杉醇耐药效果最明显。结论体外RNAi可有效沉默卵巢癌紫杉醇耐药株SKOV3/TAX细胞内stathmin基因和mdr1基因,逆转其紫杉醇耐药。
