HIV感染与差异表达的miRNA之间的相互作用
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摘要
目的探究艾滋病病毒(HIV)感染与差异表达的微小核糖核酸(miRNAs)之间的关联及机制。方法基因芯片分析3例HIV感染者、3例治疗者及2例健康对照的外周血单个核细胞(PBMC)中miRNAs表达情况并选出差异表达miRNAs,于150例样本中再次定量验证。检测转染mimic/inhibitor或过表达质粒的体外模型中p24水平改变,探究miRNAs对HIV复制的影响。通过定量聚合酶链式反应(RT-qPCR)和构建报告基因验证靶标。结果芯片筛选出3组间表达差异显著的miRNAs共11条,大部分在HIV感染者中呈下调趋势,但定量验证结果中仅hsa-miR-191-5p的差异有统计学意义(F=92.560,P<0.05)且趋势与芯片结果一致。Hsa-miR-191-5p在体外模型实验中抑制了HIV复制,后续NUP50和CCR1被证实为其靶标。结论因HIV感染引起PBMC中miRNAs差异表达,总体呈下调趋势。hsa-miR-191-5p可抑制HIV复制,可能是通过CCR1和(或)NUP50实现的。
Objective To identify relationships of HIV infection and differentially expressed miRNAs. Methods Differentially expressed miRNAs of PBMCs from HIV-1-infected patients(3 on-ART and 3 HAART-na?ve) and 2 healthy controls were screened by microarray and verified by RT-qPCR using 150 clinical samples. P24 levels of in vitro models transfected with mimic/inhibitor were detected to find the impact of miRNA on HIV. Targets predicted by bioinformatics databases and literature reports were verified by RT-qPCR and 3′UTR reporter gene. Results According to the microarray results, 11 miRNAs were found to be differentially expressed among the 3 groups, but only hsa-miR-191-5 p was significantly differentially expressed(F=92.560,P<0.05) among 150 clinical samples. Hsa-miR-191-5 p inhibited HIV replication in vitro models. NUP50 and CCR1 were found to be the targets of hsa-miR-191-5 p. Conclusion MiRNA expression is generally downregulated in PBMCs during HIV infection. HIV replication can be inhibited by hsa-miR-191-5 p by inhibiting NUP50 and/or CCR1.
Objective To identify relationships of HIV infection and differentially expressed miRNAs. Methods Differentially expressed miRNAs of PBMCs from HIV-1-infected patients(3 on-ART and 3 HAART-na?ve) and 2 healthy controls were screened by microarray and verified by RT-qPCR using 150 clinical samples. P24 levels of in vitro models transfected with mimic/inhibitor were detected to find the impact of miRNA on HIV. Targets predicted by bioinformatics databases and literature reports were verified by RT-qPCR and 3′UTR reporter gene. Results According to the microarray results, 11 miRNAs were found to be differentially expressed among the 3 groups, but only hsa-miR-191-5 p was significantly differentially expressed(F=92.560,P<0.05) among 150 clinical samples. Hsa-miR-191-5 p inhibited HIV replication in vitro models. NUP50 and CCR1 were found to be the targets of hsa-miR-191-5 p. Conclusion MiRNA expression is generally downregulated in PBMCs during HIV infection. HIV replication can be inhibited by hsa-miR-191-5 p by inhibiting NUP50 and/or CCR1.
引文
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[25] Shaw KT,Greig NH.Chemokine receptor mRNA expression at the in vitro blood-brain barrier during HIV infection[J].Neuroreport,1999,10(1):53-56.
[2] Cullen BR.MicroRNAs as mediators of viral evasion of the immune system[J].Nat Immunol,2013,14(3):205-210.
[3] Kelly EJ,Hadac EM,Cullen BR,et al.MicroRNA antagonism of the picornaviral life cycle:alternative mechanisms of interference[J].PLoS Pathog,2010,6(3):e1000820.
[4] Huang J,Wang F,Argyris E,et al.Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes[J].Nat Med,2007,13(10):1241-1247.
[5] Nathans R,Chu CY,Serquina AK,et al.Cellular microRNA and P bodies modulate host-HIV-1 interactions[J].Mol Cell,2009,34(6):696-709.
[6] Chiang K,Sung TL,Rice AP.Regulation of cyclin T1 and HIV-1 Replication by microRNAs in resting CD4+ T lymphocytes[J].J Virol,2012,86(6):3244-3252.
[7] Zhang HS,Chen XY,Wu TC,et al.MiR-34a is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation through the SIRT1/NFκB pathway[J].FEBS Lett,2012,586(23):4203-4207.
[8] Zhang HS,Wu TC,Sang WW,et al.MiR-217 is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation by down-regulation of SIRT1[J].Biochim Biophys Acta,2012,1823(5):1017-23.
[9] Swaminathan G,Navas-martin S,Martin-garcia J.MicroRNAs and HIV-1 infection:antiviral activities and beyond[J].J Mol Biol,2014,426(6):1178-1197.
[10] Pasquinelli AE.MicroRNAs and their targets:recognition,regulation and an emerging reciprocal relationship[J].Nat Rev Genet,2012,13(4):271-282.
[11] Coiras M,López-huertas MR,Pérez-olmeda M,et al.Understanding HIV-1 latency provides clues for the eradication of long-term reservoirs[J].Nat Rev Microbiol,2009,7(11):798-812.
[12] Houzet L,Yeung ML,De lame V,et al.MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1) seropositive individuals[J].Retrovirology,2008,5(118):1-9.
[13] Witwer KW,Watson AK,Blankson JN,et al.Relationships of PBMC microRNA expression,plasma viral load,and CD4+ T-cell count in HIV-1-infected elite suppressors and viremic patients[J].Retrovirology,2012,9(5):1-15.
[14] Nagpal N,Kulshreshtha R.miR-191:an emerging player in disease biology[J].Front Genet,2014,5(99):1-10.
[15] Baltimore D,Boldin MP,O′Connell RM,et al.MicroRNAs-new regulators of immune[J].Nat Immunol,2008,9(8):839-845.
[16] Hunter MP,Ismail N,Zhang X,et al.Detection of microRNA Expression in Human Peripheral Blood Microvesicles[J].PLoS One,2008,3(10):e3694.
[17] Triboulet R,Mari B,Lin YL,et al.Suppression of microRNA-silencing pathway by HIV-1 during virus replication.[J].Science,2007,315(5818):1579-82.
[18] Shen CJ,Jia YH,Tian RR,et al.Translation of Pur-α is targeted by cellular miRNAs to modulate the differentiation-dependent susceptibility of monocytes to HIV-1 infection[J].FASEB J,2012,26(11):4755-4764.
[19] Konig R,Zhou Y,Elleder D,et al.Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication[J].Cell,2008,135(1):49-60.
[20] Zhou H,Xu M,Huang Q,et al.Genome-scale RNAi screen for host factors required for HIV replication[J].Cell Host Microbe,2008,4(5):495-504.
[21] Yeung ML,Houzet L,Yedavalli VS,et al.A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication[J].J Biol Chem,2009,284(29):19463-19473.
[22] Hultquist JF,Schumann K,Woo JM,et al.A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells[J].Cell Rep,2016,17(5):1438-1452.
[23] Pollakis G,Paxton WA.Use of (alternative) coreceptors for HIV entry[J].Curr Opin HIV AIDS,2012,7(5):440-449.
[24] Chandrasekaran P,Moore V,Buckley M,et al.HIV-1 Nef down-modulates C-C and C-X-C chemokine receptors via ubiquitin and ubiquitin-independent mechanism[J].PLoS One,2014,9(1):e86998.
[25] Shaw KT,Greig NH.Chemokine receptor mRNA expression at the in vitro blood-brain barrier during HIV infection[J].Neuroreport,1999,10(1):53-56.