干扰素调节因子8在慢性阻塞性肺病中作用及其机制
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摘要
目的探讨干扰素调节因子8(IRF-8)对气道再生的影响及其机制。方法收集健康受试者和慢性阻塞性肺疾病(COPD)患者的人支气管上皮细胞(HBEC)和痰液,检测IRF8的表达并分析IRF8表达与FEV1占预计值的百分比(FEV1%)的相关性。实验分为4组:Control组(健康受试者HBEC),COPD组(COPD患者HBEC),NC组(COPD+IRF8-NC),IRF8-siRNA组(COPD+IRF8-siRNA)。分析培养上清中IL-6,IL-8和TNF-α水平,HBEC增殖能力,及p65的蛋白表达水平。荧光素酶报告实验测定IRF8的靶向调控microRNA。结果与健康对照者相比,COPD患者的HBEC和诱导痰中IRF8均显著升高。COPD患者IRF8表达水平与FEV1%呈负相关。IRF8-siRNA组IL-6,IL-8和TNF-α明显降低,细胞增殖被显著抑制,p-p65蛋白水平明显降低。同时,miR-218靶向调控IRF8的表达。结论 IRF8基因敲除能够抑制COPD患者HBEC细胞的炎症反应和细胞增殖,且miR-218靶向调控IRF8的表达。
Objective To investigate the effects of interferon regulatory factor 8(IRF8)on airway regen-eration and explore its underlying mechanism. Methods The primary human bronchial epithelial cells(HBEC),and sputum from healthy subjects and chronic obstructive pulmonary disease(COPD)patients were collected.Then the level of IRF8,and the correlation of IRF8 and FEV1% was assessed. The level of IL-6,IL-8 and TNF-αin the supernatant of medium was analyzed by ELISA. The proliferation ability of HBEC and the level of p65 wereanalyzed by MTT assay and Western blot,respectively. Luciferase reporter assay was performed to detect the targetmicroRNA of IRF8. Results IRF8 was highly increased in both the HBEC and sputum from COPD patients whencompared with that from healthy subjects. IRF8 expression was inversely correlated with FEV1% in patients withCOPD. Knockdown of IRF8 led to down-regulation of IL-6,IL-8 and TNF-α,decreased proliferation ability ofHBEC,as well as decreased level of p-p65. Importantly,miR-218 was found to be a target microRNA of IRF8.Conclusion Knockdown of IRF8 protects HBEC via a mechanism that may involve the NF-κB pathway and miR-218 regulation.
Objective To investigate the effects of interferon regulatory factor 8(IRF8)on airway regen-eration and explore its underlying mechanism. Methods The primary human bronchial epithelial cells(HBEC),and sputum from healthy subjects and chronic obstructive pulmonary disease(COPD)patients were collected.Then the level of IRF8,and the correlation of IRF8 and FEV1% was assessed. The level of IL-6,IL-8 and TNF-αin the supernatant of medium was analyzed by ELISA. The proliferation ability of HBEC and the level of p65 wereanalyzed by MTT assay and Western blot,respectively. Luciferase reporter assay was performed to detect the targetmicroRNA of IRF8. Results IRF8 was highly increased in both the HBEC and sputum from COPD patients whencompared with that from healthy subjects. IRF8 expression was inversely correlated with FEV1% in patients withCOPD. Knockdown of IRF8 led to down-regulation of IL-6,IL-8 and TNF-α,decreased proliferation ability ofHBEC,as well as decreased level of p-p65. Importantly,miR-218 was found to be a target microRNA of IRF8.Conclusion Knockdown of IRF8 protects HBEC via a mechanism that may involve the NF-κB pathway and miR-218 regulation.
引文
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[9]TAMURA T,YANAI H,SAVITSKY D,et al.The IRF family transcription factors in immunity and oncogenesis[J].Annu Rev Immunol,2008,26:535-584.
[10]SCHNEIDER D,GANESAN S,COMSTOCK A T,et al.Increased cytokine response of rhinovirus-infected airway epithelial cells in chronic obstructive pulmonary disease[J].Am JRespir Crit Care Med,2010,182(3):332-340.
[11]LIU D,CHEN Q,ZHU H,et al.Association of respiratory syncytial virus toll-like receptor 3-mediated immune response with COPD exacerbation frequency[J].Inflammation,2018,41(2):654-666.
[12]YOSHIDA Y,YOSHIMI R,YOSHII H,et al.The transcription factor IRF8 activates integrin-mediated TGF-beta signaling and promotes neuroinflammation[J].Immunity,2014,40(2):187-198.
[13]ISHII T,HOSOKI K,NIKURA Y,et al.IFN Regulatory factor3 potentiates emphysematous aggravation by lipopolysaccharide[J].J Immunol,2017,198(9):3637-3649.
[14]DI STEFANO A,CARAMORI G,GNEMMI I,et al.T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients[J].Clin Exp Immunol,2009,157(2):316-324.
[15]DI STEFANO A,CARAMORI G,OATES T,et al.Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD[J].Eur Respir J,2002,20(3):556-563.
[16]WANG I M,CONTURSI C,MASUMI A,et al.An IFN-gammainducible transcription factor,IFN consensus sequence binding protein(ICSBP),stimulates IL-12 p40 expression in macrophages[J].J Immunol,2000,165(1):271-279.
[17]PAHAN K,SHEIKH F G,LIU X,et al.Induction of nitric-oxide synthase and activation of NF-kappaB by interleukin-12 p40in microglial cells[J].J Biol Chem,2001,276(11):7899-7905.2018-04-28
[2]MANZELLA L,TIRRO E,PENNISI M S,et al.Roles of interferon regulatory factors in chronic myeloid leukemia[J].Curr Cancer Drug Targets,2016,16(7):594-605.
[3]CHMIELEWSKI S,PIASZYK-BORYCHOWSKA A,WESOLYJ,et al.STAT1 and IRF8 in vascular inflammation and cardiovascular disease:diagnostic and therapeutic potential[J].Int Rev Immunol,2016,35(5):434-454.
[4]LANGLAIS D,BARREIRO L B,GROS P.The macrophage IRF8/IRF1 regulome is required for protection against infections and is associated with chronic inflammation[J].J Exp Med,2016,213(4):585-603.
[5]CONICKX G,MESTDAGH P,AVILA COBOS F,et al.MicroRNA profiling reveals a role for microRNA-218-5p in the pathogenesis of chronic obstructive pulmonary disease[J].Am JRespir Crit Care Med,2017,195(1):43-56.
[6]SCHEMBRI F,SRIDHAR S,PERDOMO C,et al.MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium[J].Proc Natl Acad Sci U S A,2009,106(7):2319-2324.
[7]中华医学会呼吸病学分会慢性阻塞性肺疾病学组.慢性阻塞性肺疾病诊治指南(2013年修订版)[J].中华结核和呼吸杂志,2013,36(4):255-264.
[8]胡亚惠,董航明,刘来昱,等.MKK34多肽对HDM哮喘小鼠气道β-catenin表达及分布的影响[J].实用医学杂志,2017,33(4):543-547.
[9]TAMURA T,YANAI H,SAVITSKY D,et al.The IRF family transcription factors in immunity and oncogenesis[J].Annu Rev Immunol,2008,26:535-584.
[10]SCHNEIDER D,GANESAN S,COMSTOCK A T,et al.Increased cytokine response of rhinovirus-infected airway epithelial cells in chronic obstructive pulmonary disease[J].Am JRespir Crit Care Med,2010,182(3):332-340.
[11]LIU D,CHEN Q,ZHU H,et al.Association of respiratory syncytial virus toll-like receptor 3-mediated immune response with COPD exacerbation frequency[J].Inflammation,2018,41(2):654-666.
[12]YOSHIDA Y,YOSHIMI R,YOSHII H,et al.The transcription factor IRF8 activates integrin-mediated TGF-beta signaling and promotes neuroinflammation[J].Immunity,2014,40(2):187-198.
[13]ISHII T,HOSOKI K,NIKURA Y,et al.IFN Regulatory factor3 potentiates emphysematous aggravation by lipopolysaccharide[J].J Immunol,2017,198(9):3637-3649.
[14]DI STEFANO A,CARAMORI G,GNEMMI I,et al.T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients[J].Clin Exp Immunol,2009,157(2):316-324.
[15]DI STEFANO A,CARAMORI G,OATES T,et al.Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD[J].Eur Respir J,2002,20(3):556-563.
[16]WANG I M,CONTURSI C,MASUMI A,et al.An IFN-gammainducible transcription factor,IFN consensus sequence binding protein(ICSBP),stimulates IL-12 p40 expression in macrophages[J].J Immunol,2000,165(1):271-279.
[17]PAHAN K,SHEIKH F G,LIU X,et al.Induction of nitric-oxide synthase and activation of NF-kappaB by interleukin-12 p40in microglial cells[J].J Biol Chem,2001,276(11):7899-7905.2018-04-28
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