乙型肝炎病毒e抗原抑制Fas介导的肝细胞凋亡
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摘要
目的探讨乙肝病毒e抗原(HBeAg)对Fas介导的人肝癌细胞株细胞毒作用及凋亡的影响。方法构建pcDNA3.1-HBeAg重组载体并筛选表达HBeAg的肝癌细胞株,通过CCK-8、TUNEL及AnnexinⅤ实验检测HBeAg对Fas激动型抗体(anti-Fas CH11)介导的HepG2、Huh7、Hep3B细胞毒作用及凋亡的影响;过表达p53后检测HBeAg对CH11介导的Huh7、Hep3B细胞凋亡的影响;p53基因沉默或过表达后,观察HBeAg对CH11介导的HepG2细胞凋亡的影响。通过Real-time PCR和Western blot检测HBeAg对p53及mFas mRNA和蛋白表达的影响。结果 HBeAg可抑制CH11介导的HepG2细胞毒性及凋亡(均P<0.05);过表达p53后,HBeAg可抑制CH11介导的Huh7和Hep3B细胞毒性及凋亡(均P<0.05);p53的促凋亡效应可被HBeAg抑制,且靶向p53基因沉默可阻断CH11介导的细胞凋亡(均P<0.05);HBeAg可下调p53蛋白并抑制mFas转录与表达(均P<0.05)。结论 HBeAg抑制Fas介导的肝细胞凋亡依赖于p53表达,可能与下调mFas受体机制有关。
Objective To discuss the effect of hepatitis B virus e antigen(HBeAg)on Fas-mediated apoptosis of hepatoma cells.Methods pcDNA3.1-HBeAg recombinant vector was constructed,and hepatoma cells expressing HBeAg were screened.The effects of HBeAg on Fas-mediated cytotoxicity and apoptosis of hepatoma cells was tested by CCK-8,TUNEL and Annexin Ⅴ assay.The effect of HBeAg on Fas-mediated apoptosis of Huh7 and Hep3 B cells after p53 transfection was tested by Annexin Ⅴ assay.The effect of HBeAg on Fas-mediated apoptosis of HepG2 cells pretreated with p53 siRNA or overexpression was determined.The effect of HBeAg on transcription and expression of p53 and mFas was determined by real-time PCR and Western blotting.Results HBeAg inhibited CH11-mediated cytotoxicity and apoptosis of HepG2 cells(P<0.05).HBeAg inhibited CH11-mediated cytotoxicity and apoptosis of Huh7 and Hep3 B cells with p53 overexpression(P<0.05).p53-induced proapoptotic effect was inhibited by HBeAg and p53 siRNA blocked CH11-mediated apoptosis(P<0.05).HBeAg down-regulated p53 protein and inhibited transcription and expression of mFas receptor(P<0.05).Conclusion Inhibition of Fas-mediated apoptosis of hepatoma cells by HBeAg depends on p53 expression,which may be related to downregulation of mFas receptor.
Objective To discuss the effect of hepatitis B virus e antigen(HBeAg)on Fas-mediated apoptosis of hepatoma cells.Methods pcDNA3.1-HBeAg recombinant vector was constructed,and hepatoma cells expressing HBeAg were screened.The effects of HBeAg on Fas-mediated cytotoxicity and apoptosis of hepatoma cells was tested by CCK-8,TUNEL and Annexin Ⅴ assay.The effect of HBeAg on Fas-mediated apoptosis of Huh7 and Hep3 B cells after p53 transfection was tested by Annexin Ⅴ assay.The effect of HBeAg on Fas-mediated apoptosis of HepG2 cells pretreated with p53 siRNA or overexpression was determined.The effect of HBeAg on transcription and expression of p53 and mFas was determined by real-time PCR and Western blotting.Results HBeAg inhibited CH11-mediated cytotoxicity and apoptosis of HepG2 cells(P<0.05).HBeAg inhibited CH11-mediated cytotoxicity and apoptosis of Huh7 and Hep3 B cells with p53 overexpression(P<0.05).p53-induced proapoptotic effect was inhibited by HBeAg and p53 siRNA blocked CH11-mediated apoptosis(P<0.05).HBeAg down-regulated p53 protein and inhibited transcription and expression of mFas receptor(P<0.05).Conclusion Inhibition of Fas-mediated apoptosis of hepatoma cells by HBeAg depends on p53 expression,which may be related to downregulation of mFas receptor.
引文
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[19] Jing Z T,Liu W,Wu S X,et al.Hepatitis B virus surface antigen enhances the sensitivity of hepatocytes to Fas-mediated apoptosis via suppression of AKT phosphorylation[J].J Immunol,2018,201(8):2303-2314.
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[21] Jing Z T,Liu W,Xue C R,et al.AKT activator SC79 protects hepatocytes from TNF-alpha-mediated apoptosis and alleviates d-Gal/LPS-induced liver injury[J].Am J Physiol Gastrointest Liver Physiol,2019,316(3):G387-G396.
[22] Galle P R,Hofmann W J,Walczak H,et al.Involvement of the CD95(APO-1/Fas)receptor and ligand in liver damage[J].J Exp Med,1995,182(5):1223-1230.
[23] Brooks C L,Gu W.Ubiquitination,phosphorylation and ace-tylation:the molecular basis for p53 regulation[J].Curr Opin Cell Biol,2003,15(2):164-171.
[24] 刘磊,费锐,仝巧云,等.乙型肝炎病毒表面抗原调控Fas介导的肝癌细胞株凋亡[J].华中科技大学学报:医学版,2018,47(4):388-393.
[2] Dandri M,Locarnini S.New insight in the pathobiology of hepatitis B virus infection[J].Gut,2012,61(Suppl 1):16-17.
[3] Liu W,Lin Y T,Yan X L,et al.Hepatitis B virus core protein inhibits Fas-mediated apoptosis of hepatoma cells via regulation of mFas/FasL and sFas expression[J].Faseb J,2015,29(3):1113-1123.
[4] Du J,Liang X,Liu Y,et al.Hepatitis B virus core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression[J].Cell Death Differ,2009,16(2):219-229.
[5] Diao J,Khine A A,Sarangi F,et al.X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway[J].J Biol Chem,2001,276(11):8328-8340.
[6] Kondo T,Suda T,Fukuyama H,et al.Essential roles of the Fas ligand in the development of hepatitis[J].Nat Med,1997,3(4):409-413.
[7] Hayashi N,Mita E.Involvement of Fas system-mediated apoptosis in pathogenesis of viral hepatitis[J].J Viral Hepat,1999,6(5):357-365.
[8] Milich D,Liang T J.Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection[J].Hepatology,2003,38(5):1075-1086.
[9] Liu D,Cui L,Wang Y,et al.Hepatitis B e antigen and its precursors promote the progress of hepatocellular carcinoma by interacting with NUMB and decreasing p53 activity[J].Hepatology,2016,64(2):390-404.
[10] Yu Y,Wan P,Cao Y,et al.Hepatitis B virus e antigen activates the suppressor of cytokine signaling 2 to repress interferon action[J].Sci Rep,2017,7(1):1729.
[11] Wu S,Kanda T,Imazeki F,et al.Hepatitis B virus e antigen physically associates with receptor-interacting serine/threonine protein kinase 2 and regulates IL-6 gene expression[J].J Infect Dis,2012,206(3):415-420.
[12] Jegaskanda S,Ahn S H,Skinner N,et al.Downregulation of interleukin-18-mediated cell signaling and interferon gamma expression by the hepatitis B virus e antigen[J].J Virol,2014,88(18):10412-10420.
[13] Visvanathan K,Skinner N A,Thompson A J,et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein[J].Hepatology,2007,45(1):102-110.
[14] Liu S,Peng N,Xie J,et al.Human hepatitis B virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways[J].J Hepatol,2015,62(5):1015-1023.
[15] Wilson R,Warner N,Ryan K,et al.The hepatitis B e antigen suppresses IL-1 beta-mediated NF-kappaB activation in hepatocytes[J].J Viral Hepat,2011,18(10):e499-e507.
[16] Lang T,Lo C,Skinner N,et al.The hepatitis B e antigen(HBeAg)targets and suppresses activation of the toll-like receptor signaling pathway[J].J Hepatol,2011,55(4):762-769.
[17] Liu W,Jing Z T,Wu S X,et al.A novel AKT activator,SC79,prevents acute hepatic failure induced by Fas-mediated apoptosis of hepatocytes[J].Am J Pathol,2018,188(5):1171-1182.
[18] Liu W,Guo T F,Jing Z T,et al.Hepatitis B virus core protein promotes hepatocarcinogenesis by enhancing Src expression and activating the Src/PI3K/Akt pathway[J].Faseb J,2018,32(6):3033-3046.
[19] Jing Z T,Liu W,Wu S X,et al.Hepatitis B virus surface antigen enhances the sensitivity of hepatocytes to Fas-mediated apoptosis via suppression of AKT phosphorylation[J].J Immunol,2018,201(8):2303-2314.
[20] Wu S X,Chen W N,Jing Z T,et al.Hepatitis B spliced protein(HBSP)suppresses Fas-mediated hepatocyte apoptosis via activation of PI3K/Akt signaling[J].J Virol,2018,92(23):e01273-18.
[21] Jing Z T,Liu W,Xue C R,et al.AKT activator SC79 protects hepatocytes from TNF-alpha-mediated apoptosis and alleviates d-Gal/LPS-induced liver injury[J].Am J Physiol Gastrointest Liver Physiol,2019,316(3):G387-G396.
[22] Galle P R,Hofmann W J,Walczak H,et al.Involvement of the CD95(APO-1/Fas)receptor and ligand in liver damage[J].J Exp Med,1995,182(5):1223-1230.
[23] Brooks C L,Gu W.Ubiquitination,phosphorylation and ace-tylation:the molecular basis for p53 regulation[J].Curr Opin Cell Biol,2003,15(2):164-171.
[24] 刘磊,费锐,仝巧云,等.乙型肝炎病毒表面抗原调控Fas介导的肝癌细胞株凋亡[J].华中科技大学学报:医学版,2018,47(4):388-393.