羧基末端结合蛋白1对肝癌细胞HepG2增殖的影响
|
摘要
目的研究羧基末端结合蛋白1(C-terminal binding protein1,CtBP1)对人肝癌细胞HepG2增殖的影响及机制。方法采用CtBP1 siRNA转染HepG2细胞,实验分为4个组:空白对照组、转染试剂对照组、阴性序列对照组与干扰组。于转染后不同的时间收集细胞,用MTT法检测细胞增殖、流式细胞术检测细胞周期及凋亡情况。结果 CtBP1 siRNA转染有效抑制了HepG2细胞中CtBP1蛋白表达,与空白对照组比较,转染后72 h后,CtBP1蛋白下调了57.80%。CtBP1沉默后细胞生长受到抑制(P<0.05),转染后24、48、72、96 h生长抑制率分别为22.34%、52.15%、53.97%和54.77%;而对细胞周期分布无明显影响(P>0.05);但CtBP1表达下调有促凋亡作用,与对照组相比,干扰组的细胞凋亡率显著升高(P<0.05)。结论 CtBP1沉默能抑制HepG2细胞增殖,其机制是通过促凋亡来实现的。CtBP1能促进肿瘤生长和抑制凋亡,是潜在的肿瘤治疗靶点。
Obiective To investigate the impaction of C-terminal binding protein 1(CtBP1) on proliferation of hepatocellular carcinoma cell HepG2 and related mechanism.Methods siRNA targeting for CtBP1 were introduced into HepG2 cells to knock down CtBP1,3 control groups including blank control, transfection reagent and control siRNA were used as controls.At different time points after transfection, HepG2 cells were collected for MMT assay to detect cell proliferation and fl owcytometry to detect cell cycle distribution and apoptosis.Results CtBP1 siRNA effectively knocked down CtBP1 in HepG2 cell. Compared with control group, CtBP1 protein significantly was decreased by 57.80%.Cell growth were significantly inhibited after CtBP1 knockdown(P<0.05). The inhibition rates of cell growth were 22.34%,52.15%,53.97% and 54.77% at 24, 48, 72 and 96 h respectively. The distribution of cell cycle was not changed but apopotic cells were increased significantly compared with control group.Conclusion CtBP1 knockdown could inhibit cell proliferation of HepG2 mainly through apoptosis instead of cell cycle arrest. CtBP1 is involved in cell growth and apoptosis inhibition of tumor cells, and could be a candidate for cancer targeted therapy.
Obiective To investigate the impaction of C-terminal binding protein 1(CtBP1) on proliferation of hepatocellular carcinoma cell HepG2 and related mechanism.Methods siRNA targeting for CtBP1 were introduced into HepG2 cells to knock down CtBP1,3 control groups including blank control, transfection reagent and control siRNA were used as controls.At different time points after transfection, HepG2 cells were collected for MMT assay to detect cell proliferation and fl owcytometry to detect cell cycle distribution and apoptosis.Results CtBP1 siRNA effectively knocked down CtBP1 in HepG2 cell. Compared with control group, CtBP1 protein significantly was decreased by 57.80%.Cell growth were significantly inhibited after CtBP1 knockdown(P<0.05). The inhibition rates of cell growth were 22.34%,52.15%,53.97% and 54.77% at 24, 48, 72 and 96 h respectively. The distribution of cell cycle was not changed but apopotic cells were increased significantly compared with control group.Conclusion CtBP1 knockdown could inhibit cell proliferation of HepG2 mainly through apoptosis instead of cell cycle arrest. CtBP1 is involved in cell growth and apoptosis inhibition of tumor cells, and could be a candidate for cancer targeted therapy.
引文
[1]Boyd JM,Subramanian T,Schaeper U,et al.A region in the C-terminus of adenovirus 2/5 E1a protein is required for association with a cellular phosphoprotein and important for the negative modulation of T24-ras mediated transformation,tumorigenesis and metastasis[J].EMBO J,1993,12(2):469-78.
[2]Zeng SE,Xiao SJ,Zhang XL,et al.Expression of transcriptional corepressor CtBP1 and it’s target gene E-cadherin in hepatocellular carcinoma[J].Lin Chuang Yu Shi Yan Bing Li Xue Za Zhi,2010,26(6):655-7.[曾思恩,肖胜军,张小玲,等.肝细胞肝癌中转录辅抑制因子CtBP1与其靶基因E-cadherin的表达[J].临床与实验病理学杂志,2010,26(6):655-7.]
[3]Forner A,Llovet JM,Bruix J.Hepatocellular carcinoma[J].Lancet,2012,379(9822):1245-55.
[4]Llovet JM,Burroughs A,Bruix J.Hepatocellular carcinoma[J].Lancet,2003,362(9399):1907-17.
[5]Iakova P,Timchenko L,Timchenko NA.Intracellular signaling and hepatocellular carcinoma[J].Semin Cancer Biol,2011,21(1):28-34.
[6]Schaeper U,Boyd JM,Verma S,et al.Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation[J].Proc Natl Acad Sci U S A,1995,92(23):10467-71.
[7]Schaeper U,Subramanian T,Lim L,et al.Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A(CtBP)and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif[J].J Biol Chem,1998,273(15):8549-52.
[8]Bergman LM,Birts CN,Darley M,et al.CtBPs promote cell survival through the maintenance of mitotic fidelity[J].Mol Cell Biol,2009,29(16):4539-51.
[9]Bergman LM,Blaydes JP.C-terminal binding proteins:emerging roles in cell survival and tumorigenesis[J].Apoptosis,2006,11(6):879-88.
[10]Chinnadurai G.The transcriptional corepressor CtBP:a foe of multiple tumor suppressors[J].Cancer Res,2009,69(3):731-4.
[11]Mroz EA,Baird AH,Michaud WA,et al.COOH-terminal binding protein regulates expression of the p16INK4A tumor suppressor and senescence in primary human cells[J].Cancer Res,2008,68(15):6049-53.
[12]Zang JJ,Xie F,Xu JF,et al.P16 gene hypermethylation and hepatocellular carcinoma:a systematic review and metaanalysis[J].World J Gastroenterol,2011,17(25):3043-8.
[2]Zeng SE,Xiao SJ,Zhang XL,et al.Expression of transcriptional corepressor CtBP1 and it’s target gene E-cadherin in hepatocellular carcinoma[J].Lin Chuang Yu Shi Yan Bing Li Xue Za Zhi,2010,26(6):655-7.[曾思恩,肖胜军,张小玲,等.肝细胞肝癌中转录辅抑制因子CtBP1与其靶基因E-cadherin的表达[J].临床与实验病理学杂志,2010,26(6):655-7.]
[3]Forner A,Llovet JM,Bruix J.Hepatocellular carcinoma[J].Lancet,2012,379(9822):1245-55.
[4]Llovet JM,Burroughs A,Bruix J.Hepatocellular carcinoma[J].Lancet,2003,362(9399):1907-17.
[5]Iakova P,Timchenko L,Timchenko NA.Intracellular signaling and hepatocellular carcinoma[J].Semin Cancer Biol,2011,21(1):28-34.
[6]Schaeper U,Boyd JM,Verma S,et al.Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation[J].Proc Natl Acad Sci U S A,1995,92(23):10467-71.
[7]Schaeper U,Subramanian T,Lim L,et al.Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A(CtBP)and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif[J].J Biol Chem,1998,273(15):8549-52.
[8]Bergman LM,Birts CN,Darley M,et al.CtBPs promote cell survival through the maintenance of mitotic fidelity[J].Mol Cell Biol,2009,29(16):4539-51.
[9]Bergman LM,Blaydes JP.C-terminal binding proteins:emerging roles in cell survival and tumorigenesis[J].Apoptosis,2006,11(6):879-88.
[10]Chinnadurai G.The transcriptional corepressor CtBP:a foe of multiple tumor suppressors[J].Cancer Res,2009,69(3):731-4.
[11]Mroz EA,Baird AH,Michaud WA,et al.COOH-terminal binding protein regulates expression of the p16INK4A tumor suppressor and senescence in primary human cells[J].Cancer Res,2008,68(15):6049-53.
[12]Zang JJ,Xie F,Xu JF,et al.P16 gene hypermethylation and hepatocellular carcinoma:a systematic review and metaanalysis[J].World J Gastroenterol,2011,17(25):3043-8.