干扰素诱导肝癌细胞ADAR1的表达模式及HBV复制抑制
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摘要
目的探讨Ⅰ型干扰素(IFN-α、IFN-β)和Ⅱ型干扰素(IFN-γ)在肝癌细胞中诱导ADAR1(P110和P150)的表达模式及对HBV复制的作用。方法不同浓度IFN-α(0、200、500、1 000、2 000 U/mL)、IFN-β(0、200、1 000 U/mL)和IFN-γ(0、200、1 000 U/mL)分别处理肝癌细胞(Huh7和HepG2),Western blot和RT-qPCR检测肝癌细胞中IFN作用后ADAR1的表达变化;Huh7细胞瞬时转染HBV复制型质粒,IFN-α作用后,Southern blot分析HBV DNA复制中间体水平。结果Ⅰ型IFN(IFN-α、IFN-β)作用Huh7和HepG2细胞后,ADAR1 P110蛋白水平增加(P<0.05),同时ADAR1 P150表达水平明显增高(P<0.01),且ADAR1 P150的表达呈现IFN浓度依赖性;Ⅱ型IFN(IFN-γ)作用Huh7和HepG2细胞后,ADAR1 P110和P150蛋白表达水平没有发生明显变化。IFN-α明显抑制Huh7细胞中HBV复制(P<0.05),与浓度梯度呈负相关。结论Ⅰ型IFN可诱导肝癌细胞中ADAR1 P150表达,IFN-α抑制Huh7细胞中HBV复制,其作用可能与ADAR1相关。
Objective To investigate the effect of type Ⅰ interferons(IFN-α and IFN-β) and type Ⅱ interferon(IFN-γ) on the expression of adenosine deaminase acting on RNA 1(ADAR1, P110 and P150) and hepatitis B virus(HBV) replication in hepatocellular carcinoma(HCC) cells. Methods Western blotting and qRT-PCR were used to detect the expression of ADAR1 in human HCC cell lines HepG2 and Huh7 following treatment with different concentrations of IFN-α, IFN-β or IFN-γ. Southern blotting was employed to detect the changes in HBV DNA level in response to IFN-α treatment in Huh7 and HepG2 cells transfected with HBV replication plasmids. Results In Huh7 and HepG2 cells, treatments with IFN-α and IFN-β significantly increased the protein expression of ADAR1 P150 in a time-and dose-dependent manner(P<0.01) and also obviously augmented the protein level of ADAR1 P110; IFN-γ did not significantly affected the expression of either ADAR1 P110 or ADAR1 P150. IFN-α treatment dose-dependently inhibited HBV DNA replication in Huh7 cells(P<0.05). Conclusion Type Ⅰ interferons time-and dose-dependently increase the expression of ADAR1 in Huh7 and HepG2 cells, suggesting a possible role of ADAR1 in mediating the anti-HBV effect of IFN-α.
Objective To investigate the effect of type Ⅰ interferons(IFN-α and IFN-β) and type Ⅱ interferon(IFN-γ) on the expression of adenosine deaminase acting on RNA 1(ADAR1, P110 and P150) and hepatitis B virus(HBV) replication in hepatocellular carcinoma(HCC) cells. Methods Western blotting and qRT-PCR were used to detect the expression of ADAR1 in human HCC cell lines HepG2 and Huh7 following treatment with different concentrations of IFN-α, IFN-β or IFN-γ. Southern blotting was employed to detect the changes in HBV DNA level in response to IFN-α treatment in Huh7 and HepG2 cells transfected with HBV replication plasmids. Results In Huh7 and HepG2 cells, treatments with IFN-α and IFN-β significantly increased the protein expression of ADAR1 P150 in a time-and dose-dependent manner(P<0.01) and also obviously augmented the protein level of ADAR1 P110; IFN-γ did not significantly affected the expression of either ADAR1 P110 or ADAR1 P150. IFN-α treatment dose-dependently inhibited HBV DNA replication in Huh7 cells(P<0.05). Conclusion Type Ⅰ interferons time-and dose-dependently increase the expression of ADAR1 in Huh7 and HepG2 cells, suggesting a possible role of ADAR1 in mediating the anti-HBV effect of IFN-α.
引文
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[25] 李京源,李涛,朱席琳,等.ADAR1通过RNA编辑上调ZNF655表达并促进人肝癌细胞系HepG2中HBV复制[J].基础医学与临床,2018,38(3):312-316.DOI:10.16352/j.issn.1001-6325.2018.03.006.LI J Y,LI T,ZHU X L,et al.ADAR1 up-regulates ZNF655 expression via RNA editing and enhances HBV replication in HepG2 cell line[J].Basic Clin Med,2018,38(3):312-316.DOI:10.16352/j.issn.1001-6325.2018.03.006.
[26] LI L,QIAN G,ZUO Y,et al.Ubiquitin-dependent turnover of adar1 is required for efficient antiviral activity of type Ⅰ interferon[J].J Biol Chem,2016,291(48):24974-24985.DOI:10.1074/jbc.M116.737098.
[2] TRéPO C,CHAN H L,LOK A.Hepatitis B virus infection[J].Lancet,2014,384(9959):2053-2063.DOI:10.1016/s0140-6736(14)60220-8.
[3] SAMUEL C E.Adenosine deaminases acting on RNA (ADARs) are both antiviral and proviral[J].Virology,2011,411(2):180-193.DOI:10.1016/j.virol.2010.12.004.
[4] CHAN T H,LIN C H,QI L,et al.A disrupted RNA editing balance mediated by ADARs (Adenosine Deaminases that act on RNA) in human hepatocellular carcinoma[J].Gut,2014,63(5):832-843.DOI:10.1136/gutjnl-2012-304037.
[5] LI Z,OKONSKI K M,SAMUEL C E.Adenosine deaminase acting on RNA 1 (ADAR1) suppresses the induction of interferon by measles virus[J].J Virol,2012,86(7):3787-3794.DOI:10.1128/JVI.06307-11.
[6] LI Z,WOLFF K C,SAMUEL C E.RNA adenosine deaminase ADAR1 deficiency leads to increased activation of protein kinase PKR and reduced vesicular stomatitis virus growth following interferon treatment[J].Virology,2010,396(2):316-322.DOI:10.1016/j.virol.2009.10.026.
[7] CROAGH C M,DESMOND P V,BELL S J.Genotypes and viral variants in chronic hepatitis B:A review of epidemiology and clinical relevance[J].World J Hepatol,2015,7(3):289-303.DOI:10.4254/wjh.v7.i3.289.
[8] SATO S,LI K,KAMEYAMA T,et al.The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus[J].Immunity,2015,42(1):123-132.DOI:10.1016/j.immuni.2014.12.016.
[9] LV M,ZHANG B,SHI Y,et al.Identification of BST-2/tetherin-induced hepatitis B virus restriction and hepatocyte-specific BST-2 inactivation[J].Sci Rep,2015,5:11736.DOI:10.1038/srep11736.
[10] YAN R,ZHAO X,CAI D,et al.The interferon-inducible protein tetherin inhibits hepatitis B virus virion secretion[J].J Virol,2015,89(18):9200-9212.DOI:10.1128/JVI.00933-15.
[11] LEONG C R,FUNAMI K,OSHIUMI H,et al.Interferon-stimulated gene of 20 kDa protein (ISG20) degrades RNA of hepatitis B virus to impede the replication of HBV in vitro and in vivo[J].Oncotarget,2016,7(42):68179-68193.DOI:10.18632/oncotarget.11907.
[12] LIU Y,NIE H,MAO R,et al.Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA[J].PLoS Pathog,2017,13(4):e1006296.DOI:10.1371/journal.ppat.1006296.
[13] TAN G,XIAO Q,SONG H,et al.Type I IFN augments IL-27-dependent TRIM25 expression to inhibit HBV replication[J].Cell Mol Immunol,2018,15(3):272-281.DOI:10.1038/cmi.2016.67.
[14] CHEN Y,HU J,CAI X,et al.APOBEC3B edits HBV DNA and inhibits HBV replication during reverse transcription[J].Antiviral Res,2018,149:16-25.DOI:10.1016/j.antiviral.2017.11.006.
[15] BONVIN M,ACHERMANN F,GREEVE I,et al.Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication[J].Hepatology,2006,43(6):1364-1374.DOI:10.1002/hep.21187.
[16] HU J,QIAO M,CHEN Y,et al.Cyclin E2-CDK2 mediate SAMHD1 phosphorylation to abrogate its restriction of HBV replication in hepatoma cells[J].FEBS Lett,2018,592(11):1893-1904.DOI:10.1002/1873-3468.13105.
[17] JEONG G U,PARK I H,AHN K,et al.Inhibition of hepatitis B virus replication by a dNTPase-dependent function of the host restriction factor SAMHD1[J].Virology,2016,495:71-78.DOI:10.1016/j.virol.2016.05.001.
[18] LI L,LEI Q S,ZHANG S J,et al.suppression of USP18 potentiates the anti-HBV activity of interferon alpha in HepG2.2.15 cells via JAK/STAT signaling[J].PLoS ONE,2016,11(5):e0156496.DOI:10.1371/journal.pone.0156496.
[19] LI Y,LI S,DUAN X.et al.Interferon-stimulated gene 15 conjugation stimulates hepatitis B virus production independent of type I interferon signaling pathway in vitro [J].Mediators Inflamm,2016,2016:7417648.DOI:10.1155/2016/7417648
[20] KOEBERLEIN B,ZUR HAUSEN A,BEKTAS N,et al.Hepatitis B virus overexpresses suppressor of cytokine signaling-3 (SOCS3) thereby contributing to severity of inflammation in the liver[J].Virus Res,2010,148(1/2):51-59.DOI:10.1016/j.virusres.2009.12.003.
[21] 郜衍周,范红霞,邱立鹏,等.IFN-α对gp96的上调降低其抗HBV的效率[J].微生物学报,2013,53(8):867-874.HAO Y Z,FAN H X,QIU L P.et al IFN-α-induced gp96 upregulation negatively affects the anti-HBV efficiency of IFN-α [J].Acta Microbiol Sinica,2013,53(8):867-874.
[22] XIAO C,QIN B,CHEN L,et al.Preactivation of the interferon signalling in liver is correlated with nonresponse to interferon alpha therapy in patients chronically infected with hepatitis B virus[J].J Viral Hepat,2012,19(2):e1-e10.DOI:10.1111/j.1365-2893.2011.01471.x.
[23] HAN M,LI Y,WU W,et al.Altered expression of interferon-stimulated genes is strongly associated with therapeutic outcomes in hepatitis B virus infection[J].Antiviral Res,2017,147:75-85.DOI:10.1016/j.antiviral.2017.10.003.
[24] WU X,SHI W,WU J,et al.A functional polymorphism in ADAR 1 gene affects HBsAg seroclearance both spontaneously and interferon induced[J].Liver Int,2014,34(10):1560-1565.DOI:10.1111/liv.12444.
[25] 李京源,李涛,朱席琳,等.ADAR1通过RNA编辑上调ZNF655表达并促进人肝癌细胞系HepG2中HBV复制[J].基础医学与临床,2018,38(3):312-316.DOI:10.16352/j.issn.1001-6325.2018.03.006.LI J Y,LI T,ZHU X L,et al.ADAR1 up-regulates ZNF655 expression via RNA editing and enhances HBV replication in HepG2 cell line[J].Basic Clin Med,2018,38(3):312-316.DOI:10.16352/j.issn.1001-6325.2018.03.006.
[26] LI L,QIAN G,ZUO Y,et al.Ubiquitin-dependent turnover of adar1 is required for efficient antiviral activity of type Ⅰ interferon[J].J Biol Chem,2016,291(48):24974-24985.DOI:10.1074/jbc.M116.737098.