MicroRNA-155对3种可经输血传播RNA病毒复制的影响及机制探究
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摘要
目的了解经血传播的RNA病毒感染细胞后对小分子非编码RNA-155 (miR-155)的诱导作用及miR-155对这类病毒复制的影响。方法在2×10~5/mL肝癌细胞系Huh7.5.1中分别感染3种RNA病毒HCV、DENV和ZIKV,感染复数(MOI)为0.2,感染后48 h,提取胞内RNA,通过荧光定量PCR (qRT-PCR)的方法,检测病毒感染后miR-155的相对表达变化;同时在2×10~5/mL Huh7.5.1细胞中转染10 pmol的miR-155模拟物(miR-155 mimic)使miR-155高表达,转染后12 h分别接种0.2 MOI的3种病毒,感染后48 h提取胞内RNA,通过荧光定量PCR检测3种病毒RNA的复制水平及宿主抗病毒先天免疫相关基因表达水平。结果与未感染病毒的Huh7.5.1细胞相比,HCV、DENV及ZIKV感染Huh7.5.1细胞48 h后,miR-155的相对表达分别升高了132倍、157倍和8倍。与转染阴性对照组相比,miR-155高表达的Huh7.5.1细胞中,HCV、ZIKV和DENV的复制分别降低了48%、54%和70%;miR-155高表达的Huh7.5.1细胞中,IFNβ及一些干扰素刺激基因的表达明显升高。结论 HCV、DENV和ZIKV感染Huh7.5.1细胞后,显著诱导细胞中miR-155的表达;miR-155在细胞中高表达后,通过调节宿主细胞中Ⅰ型干扰素及一些干扰素刺激基因的表达来抑制HCV、DENV和ZIKV的复制。
Objective To investigate the induction of three kinds of blood transfusion transmitted RNA virus on miR-155 and the role of miR-155 in HCV, DENV and ZIKV replication. Methods Huh7.5.1 cells were seeded on a 12-well plate at 2×10~5/mL and infected with HCV, DENV and ZIKV at 0.2 MOI, respectively. The cell RNAs 48 hours after infection were harvested and the relative expression of miR-155 was tested using qRT-PCR. Meanwhile, Huh7.5.1 cells were seeded on a 12-well plate at 2×10~5/mL and transfected miR-155 mimic at 10 pmol/mL 12 h before virus infection, and RNA was harvested 48 h post-infection and relative gene mRNA was tested using qRT-PCR. Results The relative expression of miR-155 increased significantly by 132 folds, 157 folds and 8 folds in HCV, DENV and ZIKV infected Huh7.5.1 cells, respectively. And miR-155 overexpression inhibited HCV, ZIKV and DENV replication by about 48%, 54% and 70%, respectively compared with the negative mimic transfection cells. MiR-155 overexpression upregulated IFNβ and its downstream interferon stimulated genes(ISGs) expression. Conclusion HCV, DENV and ZIKV infection induce miR-155 expression in Huh7.5.1 cells; miR-155 inhibits HCV, DENV and ZIKV replication through regulating IFN pathway and ISGs expression.
Objective To investigate the induction of three kinds of blood transfusion transmitted RNA virus on miR-155 and the role of miR-155 in HCV, DENV and ZIKV replication. Methods Huh7.5.1 cells were seeded on a 12-well plate at 2×10~5/mL and infected with HCV, DENV and ZIKV at 0.2 MOI, respectively. The cell RNAs 48 hours after infection were harvested and the relative expression of miR-155 was tested using qRT-PCR. Meanwhile, Huh7.5.1 cells were seeded on a 12-well plate at 2×10~5/mL and transfected miR-155 mimic at 10 pmol/mL 12 h before virus infection, and RNA was harvested 48 h post-infection and relative gene mRNA was tested using qRT-PCR. Results The relative expression of miR-155 increased significantly by 132 folds, 157 folds and 8 folds in HCV, DENV and ZIKV infected Huh7.5.1 cells, respectively. And miR-155 overexpression inhibited HCV, ZIKV and DENV replication by about 48%, 54% and 70%, respectively compared with the negative mimic transfection cells. MiR-155 overexpression upregulated IFNβ and its downstream interferon stimulated genes(ISGs) expression. Conclusion HCV, DENV and ZIKV infection induce miR-155 expression in Huh7.5.1 cells; miR-155 inhibits HCV, DENV and ZIKV replication through regulating IFN pathway and ISGs expression.
引文
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[2] Young PR.Arboviruses:a family on the move.Adv Exp Med Biol,2018,1062(1):1-10.
[3] Rodriguez A,Vigorito E,Clare S,et al.Requirement of bic/microRNA-155 for normal immune function.Science,2007,316 (5824):608-611.
[4] Yin Q,McBride J,Fewell C,et al.MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways.J Virol,2008,82 (11):5295-5306.
[5] Dickey LL,Hanley TM,Huffaker TB,et al.MicroRNA 155 and viral-induced neuroinflammation.J Neuroimmunol,2017,308(1):17-24.
[6] Zhang Y,Wei W,Cheng N,et al.Hepatitis C virus-induced up-regulation of microRNA-155 promotes hepatocarcinogenesis by activating Wnt signaling.Hepatology,2012,56 (5):1631-1640.
[7] El-Ekiaby N,Hamdi N,Negm M,et al.Repressed induction of interferon-related microRNAs miR-146a and miR-155 in peripheral blood mononuclear cells infected with HCV genotype 4.FEBS Open Bio,2012,2:179-186.
[8] Swaminathan G,Rossi F,Sierra LJ,et al.A role for microRNA-155 modulation in the anti-HIV-1 effects of Toll-like receptor 3 stimulation in macrophages.PLoS Pathog,2012,8 (9):e1002937.
[9] Verdonck K,Gonzalez E,Van Dooren S,et al.Human T-lymphotropic virus 1:recent knowledge about an ancient infection.Lancet Infect Dis,2007,7 (4):266-281.
[10] Su C,Hou Z,Zhang C,et al.Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells.Virol J,2011,8:354.
[11] Taganov KD,Boldin MP,Chang KJ,et al.NF-kappaB-dependent induction of microRNA miR-146,an inhibitor targeted to signaling proteins of innate immune responses.Proc Natl Acad Sci U S A,2006,103 (33):12481-12486.
[12] Mobergslien A,Sioud M.Exosome-derived miRNAs and cellular miRNAs activate innate immunity.J Innate Immun,2014,6 (1):105-110.
[13] Hei L,Zhong J.Laboratory of genetics and physiology 2 (LGP2) plays an essential role in hepatitis C virus infection-induced interferon responses.Hepatology,2017,65 (5):1478-1491.
[14] Bhela S,Mulik S,Reddy PB,et al.Critical role of microRNA-155 in herpes simplex encephalitis.J Immunol,2014,192 (6):2734-2743.
[15] Zhai A,Qian J,Kao W,et al.Borna disease virus encoded phosphoprotein inhibits host innate immunity by regulating miR-155.Antiviral Res,2013,98 (1):66-75.
[16] Vigorito E,Perks KL,Abreu-Goodger C,et al.microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells.Immunity,2007,27 (6):847-859.
[17] Rothchild AC,Sissons JR,Shafiani S,et al.MiR-155-regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis.Proc Natl Acad Sci USA,2016,113 (41):E6172-E6181.