| Chronic obstructive pulmonary disease(COPD)is a chronic heterogeneous disease of the lungs characterised by persistent and excessive inflammation,leading to tissue remodelling,alveolar lesions,airflow limitation and accelerated lung function decline.Cigarette smoking is the predominant risk factor for COPD.Inhaled cigarette smoke first encounters the airway epithelium,which causes the release of pro-inflammatory mediators,including damage-associated molecular cytokines like interleukin(IL)-6,IL-8 and tumour necrosis factor(TNF)-?.Inflammatory cytokines cause lung infiltration by inflammatory cells that further amplify the inflammation and release proteases and reactive oxidative species that damage the parenchymal lung tissue,contributing to emphysema development in COPD patients.Injured lung epithelial cells have the potential to change the finely balanced reciprocal interaction between the lung epithelial and subepithelial fibroblasts,which leads to fibroblast activation,and causes excessive extracellular matrix deposition and remodelling of the small airways in COPD.Although inflammation-based airway injury is a well known central feature of COPD,the detail molecular mechanisms are not fully understood.Furthermore,there is no cure for COPD and current medication cannot reverse the long-term decline in lung function because of its heterogeneous.Micro RNA(miRNA),characterized as single stranded and 22 nucleotides in length,are small non-coding RNA that post-transcriptionally regulate the gene expression by inducing m RNA degradation or inhibition of translation.Additionally,it is now well accepted that miRNAs do not only act in one particular cell type,but are actively transported within extracellular vesicles(EV).Mi RNAs are now recognized as major regulators in physiological homeostasis and disease pathogenesis in the airway microenvironment.Their importance in lung pathology has prompted the development of miRNA-based therapies and biomarker tools.Although cigarette smoke has been shown to alter miRNA levels in lung,and could be implicated in COPD,the key functions of miRNA in the disease pathogenesis is lacking.Therefore,characterizing the roles of miRNAs in the dysfunction of airway epithelial cells by exposure to CS and in the aberrant communication between epithelial cells and sub-epithelial fibroblasts,will be important to understand the mechanism of COPD pathogenesis and be help to indentificate the early biological markers and novel therapeutic targets.Part ? The roles of miR-218 in CSE-induced inflammatory response and mucus hyper-production in airway bronchial cellsObjective To investigate the role of miR-218,acting via TNFR1/NF-?B,in airway inflammation and mucin hyper-secretion induced by cigarette smoking.Methods A cell model of human bronchial epithelial(HBE)cells exposed with cigarette smoke extract(CSE)were mimic the cigarette smoke exposure.q RT-PCR was used to detect the expression of miR-218,IL-6,and IL-8.Enzyme-Linked Immunosorbent Assays(ELISA)was used to determine the levels of IL-6,IL-8,and MUC5 AC in cell media.Western blots was performed to determine the expression of MUC5 AC,TNFR1,and p-p65.Luciferase reporter assay was investigated the combination of miR-218 and TNFR1.To confirm miR-218-mediated TNFR1 is involved in CSE-induced production of MUC5 AC and proinflammatory cytokines,HBE cells were transduced with miR-218 inhibitor/mimic,TNFR1 si RNA,or CRISP/Cas9 SAM,or pretreated with Andrographolide(Andro)and then exposed to CSE,respectively.Results1.The roles of miR-218 in CSE-induced decrease of miR-218 levels,mucus hyper-production and inflammatory response in HBE cells.HBE cells were exposed to 20 ?g/mL CSE for 0,12,24 or 48 h,respectively.The results showed that after treatment with CSE,the levels of miR-218 were markedly decreased,whereas the expression and secretion levels of IL-6,IL-8,and MUC5 AC were significant increased in a time-dependent manner.Suggesting that in HBE cells,CSE decreases the miR-218 levels,increases the expression and secretion of MUC5 AC and inflammatory cytokines.HBE cells were transfected for 24 h with the miR-218 mimic or miR-NC mimic(50 n M),then incubated with CSE(20 ?g/m L)for 24 h.The results showed that up-regulation of miR-218 by the mimic reduced the CSE-induced increases of IL-6,IL-8,and MUC5 AC,suggesting that miR-218 is involved in the CSE-induced mucus hyper-production and inflammatory response.2.The roles of miR-218 in CSE-induced the activation of TNFR1/NF-?B in HBE cellsHBE cells were exposed to 20 ?g/mL CSE for 0,12,24 or 48 h,respectively.Compared with cells not exposed to CSE,incubation of HBE cells with CSE resulted in a significant increase of the protein levels of TNFR1 and p-p65 in a time-dependent manner.Suggesting that CSE activation of TNFR1/NF-?B signaling.HBE cells were transfected for 24 h with the miR-218 mimic or miR-NC mimic(50 n M),then incubated with CSE(20 ?g/m L)for 24 h.The results showed that up-regulation of miR-218 by the mimic reduced the CSE-induced increases of TNFR1 and p-p65,suggesting that miR-218 is involved in the CSE-induced activation of TNFR1/NF-?B signaling.Bioinformatics analysis revealed 3'UTRs of TNFR1 that contain putative miR-218 interacting regions.HBE cells were co-transfected for 48 h with the miR-218 mimic or miR-NC mimic(20 n M)and the TNFR1-wild or mutant plasmid(1 ?g).Luciferase assays revealed that transfection of cells with the miR-218 mimic inhibited the luciferase activity of TNFR1 3'UTR,but the TNFR1 mutant showed no response to the mimic,indicating that,in HBE cells,miR-218 binds to the 3'UTR of TNFR1 and inhibits CSE-induced activation of NF-?B signaling.3.The roles of TNFR1/NF-?B in CSE-induced mucus hyper-production and inflammatory response in HBE cellsHBE cells were transfected for 24 h with TNFR1 si RNA or control si RNA(20 n M),then incubated with CSE(0 or 20 ?g/m L)for 24 h.Down-regulation of TNFR1 blocked the CSE-induced increase of levels of p-p65,MUC5 AC,IL-6,and IL-8,suggesting that TNFR1 is involved in CSE-induced NF-?B activation,mucus hyper-production and inflammatory response in HBE cells.4.The roles of miR-218-mediated TNFR1 in CSE-induced mucus hyper-production and inflammatory responseHBE cells were co-transfected for 24 h with the miR-218 mimic or miR-NC mimic(50 n M)and TNFR1 SAM or control SAM(2 ?g),then incubated with CSE(0 or 20 ?g/m L)for 24 h.In CSE-treated cells transfected with the miR-218 mimic,there were lower levels of p-p65,IL-6 and IL-8,but the levels were restored in CSE-treated cells co-transfected with the miR-218 mimic and TNFR1 SAM.These data provide evidence that,in HBE cells,miR-218 is involved in CSE-induced activation of NF-?B,mucus hyper-production and inflammatory response through mediating TNFR1.5.The roles of miR-218-mediated TNFR1/NF-?B in andrographolide protective against CSE-induced inflammatory response and mucus hyper-production in HBE cellsHBE cells were pre-treated for 12 h with DMSO(0.05%)or Andro(5?M),then incubated with CSE(20 ?g/m L)for 24 h.The results showed that the pre-treatment with andro caused a significant restoration of the expression of miR-218,but reduction in the expression of IL-6,IL-8,MUC5 AC,TNFR1 and p-p65 in CSE-exposed HBE cells,suggesting that Andro can block CSE-induced downregulation of miR-218,activation of TNFR1/NF-?B,mucus hyper-production and inflammatory response in HBE cells.After transfection of miR-218 inhibitor or miR-NC inhibitor(50 nM)for 12 h,HBE cells were pretreated for 12 h with Andro(5 ?M)or DMSO(0.05%),then incubated with CSE(0 or 20 ?g/m L)for 24 h.In CSE-treated cells pretreated with Andro,there were lower levels of TNFR1,p-p65,IL-6,IL-8,and MUC5 AC,but the levels were restored in CSE-treated cells co-incubated with the miR-218 inhibitor and Andro.These data provide evidence that silencing of miR-218 attenuates the negative mediation of Andro on CSE-induced TNFR1/NF-?B activation,mucus hyper-production and inflammatory response in HBE cells.Conclusion In HBE cells,CSE induces the inflammatory response and mucus hyper-production.CSE induces the decreases of miR-218 and increase of TNFR1 and p-p65 levels.miR-218 acts by repressing TNFR1-mediated activation of NF-?B,which is involved in mucus hyper-production and inflammatory response induced by CSE.Andro antagonized CSE-induced inflammatory response and mucus hyper-production in through induction of miR-218.Part ? The roles of miR-21 in CSE-induced differentiation of airway fibroblast to myofibroblastObjective To investigate the role and mechanism of exosomal miR-21 derived from bronchial epithelial cells in the regulation of myofibroblast differentiation induced by cigarette smoking through the p VHL/HIF-1? signaling pathway.Methods An in vitro model was established using a co-culturing model in which HBE cells were co-cultured with fetal lung fibroblasts(MRC-5)or MRC-5 cells were treated with exosomes derived from HBE cells exposed to CSE.The morphology of the exosomes was investigated by the transmission electron microscope.The protein markers of exosomes were determined by western blots.The levels of miR-21 in cells and exosomes were detected by q RT-PCR.The levels of p VHL,HIF-1?,?-SMA and Collagen I in the MRC-5 cells were determined by western blots.The combination of miR-21 and the 3'UTR of pVHL was detected by luciferase reporter assay.GW4869(an inhibitor of the exosome synthesis),miR-21 inhibitor or mimic,p VHL si RNA were used to investigate the role of the exosomal miR-21 derived from bronchial epithelial cells in the regulation of myofibroblast differentiation in MRC-5 cells induced by CSE through the p VHL/HIF-1? signaling pathway.Results1.The roles of exosomes derived from CSE-treated HBE cells in transferring miR-21 to MRC-5 cellsExosomes were isolated after HBE cells were exposed to 0 or 20 ?g/mL CSE for 12 h.Vesicles that match the characteristics of exosomes could be detected in both HBE cells and CSE-exposed HBE cells.Exosomes from normal(HBE-Exo)and CSE-treated HBE cells(CHBE-Exo)were added to MRC-5 cells.After 3 h,these cells exhibited uptake of exosomes,indicating the internalization of PKH76-labeled exosomes.These results indicate that both normal and CSE-treated HBE cells can secrete exosomes which can be up taken by MRC-5 cells.Exosomes were isolated after HBE cells were exposed to 0 or 20 ?g/mL CSE for 12 h.miR-21 levels were increased in CSE-treated HBE cells and its released exosomes.These results indicate that CSE can induce the increase of levels of miR-21 in HBE cells and transferred to extracellular by exosomes.MRC-5 cells were exposed to 50 ?g/mL HBE-Exo or CHBE-Exo for 24 h,or co-cultured with HBE cells or CSE pretreated HBE(CHBE)cells,respectively.Mi R-21 levels were increased in MRC-5 cells exposed to CHBE-Exo as well as co-cultured with CHBE cells.Further,the elevated levels of miR-21 in MRC-5 cells co-cultured with CHBE cells were prevented by GW4869.These results suggest that CSE-induced the increase of levels of miR-21 in HBE cells can transfer into MRC-5 cells by exosomes.2.The roles of exosomal miR-21 derived from CSE-treated HBE cells in the myofibroblast differentiation phenotypeMRC-5 cells were exposed to 50 ?g/m L HBE-Exo or CHBE-Exo for 24 h,or co-cultured with HBE or CHBE cells for 24 h,respectively.The levels of ?-SMA and Collagen I were significantly increased in MRC-5 cells exposed to CHBE-Exo as well as co-cultured with CHBE cells.In addition,the increases of ?-SMA and collagen I expression in MRC-5 cells were blocked by GW4869.These results suggest that exosomes derived from HBE cells can promote the myofibroblast differentiation of MRC-5 cells.HBE cells were transfected for 24 h with the miR-21 inhibitor or miR-NC inhibitor(50 n M),then incubated with CSE(20 ?g/m L)for 12 h.The levels of miR-21 in exosomes derived from HBE cells transfected with a miR-21 inhibitor and treated with CSE(anti-miR-21-CHBE-Exo)significantly decreased.Further,the levels of ?-SMA and collagen I in MRC-5 cells treated with anti-miR-21-CHBE-Exo also significantly decreased.These results indicate that the exosomal miR-21 derived from CSE-treated HBE cells play an important role in the myofibroblast differentiation of MRC-5 cells.3.Effects of the exosomal miR-21 derived from CSE-treated HBE cells on the p VHL/HIF-1? pathway in MRC-5 cellsMRC-5 cells were exposed to 50 ?g/mL HBE-Exo or CHBE-Exo for 24 h,respectively.The level of p VHL in MRC-5 cells treated with CHBE-Exo decreased while the level of HIF-1? increased.These results indicate that exosomes derived from CSE-treated HBE cells can affect the expression of p VHL and HIF-1? in MRC-5 cells.By use of two independent online databases,we predicted that miR-21 regulates p VHL by binding to p VHL 3' UTR regions.Following co-transfection of MRC-5 cells with a plasmid containing wild-type p VHL 3'UTR and a miR-21 mimic,there was low luciferase activity;however,co-transfection with a miR-21 mimic with mutated forms of p VHL 3'UTR resulted in no appreciable change in luciferase activity.Consistently,exosomes derived from CSE-treated HBE cells also decreased the luciferase activity of p VHL 3'UTR,but not that for mutant p VHL 3'UTR.These observations indicate that,in MRC-5 cells,exosomal miR-21 derived from CSE-treated HBE cells binds to the 3'UTR of p VHL m RNA and inhibits p VHL expression.Down-regulation of exosomal miR-21 using a miR-21 inhibitor blocked the changes of p VHL and HIF-1? levels caused by exosomes derived from CSE-treated HBE cells but knockdown of p VHL attenuated the effect of the miR-21 inhibitor.These results show that exosomal miR-21 derived from CSE-treated HBE cells regulate the expression of HIF-1? in MRC-5 cells via p VHL.4.Effects of exosomal miR-21 derived from CSE-treated HBE cells regulating HIF-1? via p VHL in the myofibroblast differentiationDown-regulation of exosomal miR-21 after knockdown of pVHL in MRC-5 cells evaluated the levels of ?-SMA and Collagen I significantly.These results indicate that exosomal miR-21 derived from CSE-treated HBE cells regulates the myofibroblast differentiation of MRC-5 cells via p VHL.Down-regulation of HIF-1? in MRC-5 cells treated with CHBE-Exo reduced the levels of HIF-1???-SMA and Collagen I which indicate that HIF-1? plays an important role in the myofibroblast differentiation of MRC-5 cells induced by exosomes derived from CSE-treated HBE cells.Conclusion CSE can induce the up-regulation of miR-21 in HBE cells which can be transferred into MRC-5 cells.Exosomal miR-21 plays an important role in inducing the activation and differentiation of MRC-5 cells regulated by HIF-1? via p VHL.Part ? The roles of miR-218 and miR-21 in cigarette smokeinduced airway injury in miceObjective Investigate the roles of miR-218 in Andrographolide protective against cigarette smoke(CS)-induced airway inflammation and mucin hyper-secretion.Besides,investigate the protective effects of miR-21 silencing in CS-induced airway remodeling.Methods Male BALB/c mice at 6-8 weeks of age were random divided into four groups,control(Con),cigarette smoke(CS),CS plus Andro,and CS plus NS,and then exposed to CS(500 mg/m3 total particulate matter,TPM)in a whole-body exposure system for 4 weeks.Andro and NS were administered by Intraperitoneal injection.Bronchoalveolar lavage fluid(BALF)assays were performed to determine the inflammation levels.q RT-PCR was used to detect the expression of miR-218,IL-6,and IL-8 in lung tissue.Western blots was performed to determine the expression of MUC5 AC,TNFR1,and p-p65 in lung tissue.ELISA was used to determine the levels of IL-6,IL-8,and MUC5 AC in BALF.6-8-weeks-old male BALB/c mice were selected to establish an animal model of mice with airway injury induced by CS by randomly divided into Con group,CS group,CS plus anti-miR-21 group and CS plus anti-miR-NC group.The control group was exposed to air while the other groups were exposed to 500 mg/m3 TPM CS for 8 weeks.Antagomir-21 and an antagomir negative control were administrated by tail vein injection.Lung function was determined by whole-body plethysmography.The fibrosis of collagen was detected by Masson Trichrome.The levels of miR-21 in lung tissues were determined by q RT-PCR.The protein levels of p VHL,HIF-1?,?-SMA and Collagen I were detected by western blots.Results1.The effects of CS on airway inflammatory response,mucin secretion,the expression of miR-218 and TNFR1/NF-?B in miceMice were random divided into groups of Con or CS,and then exposed to Air or CS for 4 weeks.The results showed that a significant increase in total cell number in the BALF was found in mice exposed to CS,compared with controls.The inflammatory infiltrate,characterized by a significant increase in mononuclear cells and neutrophils,was also observed in CS-exposed mice.The levels of IL-6,IL-8,and MUC5 AC were significant increase in lung tissue and BALF in CS-exposed mice.Furthermore,the levels of miR-218 was downregulated,but the protein levels of TNFR1 and p-p65 were upregulated in lungs of mice exposed to CS.Suggesting that CS exposure increases airway inflammatory response,mucin secretion,reduces miR-218 levels and activates TNFR1/NF-?B signaling in mice.2.The effects of Andro on CS-induced airway inflammatory response,mucin secretion,the decrease of miR-218 levels and TNFR1/NF-?B activation in miceMice were random divided into 4 groups,including Con,CS,CS plus Andro,and CS plus NS,and then exposed to Air or CS for 4 weeks.The results showed that,in CS-exposed mice,Andro attenuated the immune cells counts and decreased IL-6,IL-8,and MUC5 AC levels in airway;the expression of IL-6,IL-8,and MUC5 AC levels were decreased in lung tissue.Furthermore,treatment with andro caused a significant restoration of the expression of miR-218,but reduction in the expression of TNFR1 and p-p65 in lung of CS-exposed mice.Suggesting that Andro can block CS-induced inflammatory response,mucin secretion,downregulation of miR-218 and TNFR1/NF-?B activation in mice.3.Effects of CS exposure on the function and structure of airway,and the levels of miR-21 and p VHL/HIF-1? in MiceMice were random divided into groups of Con or CS,and then exposed to Air or CS for 8 weeks.The mice,exposed to CS,developed airway remodeling and an emphysematous phenotype,showing augmented airway hyperresponsiveness(AHR),and airway thickening and collagen deposition and the levels of ?-SMA and Collagen I in lung tissue were significantly increased.Moreover,the levels of miR-21 and HIF-1? were upregulated while the level of p VHL was downregulated in lungs of mice exposed to CS compared to the control.The results indicate that exposure to CS can induce decline of lung function and airway fibrosis,and can upregulate the level of miR-21 and activate the p VHL/HIF-1? pathway in lungs of mice.4.The roles of miR-21 on CS-induced the aberrant lung function and structure of airway,and the activation of p VHL/HIF-1? in miceMice were random divided into 4 groups,including Con,CS,CS plus Andro,and CS plus NS,and then exposed to Air or CS for 8 weeks.In CS-exposed mice,downregulation of miR-21 prevented the changes in pulmonary function and attenuated airway obstruction.Also,the expression of ?-SMA and Collagen I levels were decreased.Moreover,the expression of p VHL was restored in the group mice treated with antagomir-21,whereas HIF-1? levels were decreased as well as miR-21 levles.These results show that downregulation of miR-21 prevents CS-induced airway injury and p VHL/HIF-1? activation in mice.Conclusion Eposure of CS for 4 weeks can induce airway inflammation and mucin hypersecretion,and induced the decrease of miR-218 levels,and increase of TNFR1 and p-p65 expression in lung of mice.Andro restored CS-induced decrease of miR-218 levels,and prevented CS-induced inflammation and mucin hyper-secretion in lung of mice.Exposure to CS for 8 weeks can induce airway injury in mice as well as the up-regulated level of miR-21 in lung tissues and the activation of p VHL/HIF-1? pathway.Downregulation of miR-21 prevents CS-induced airway injury in mice.Part ? The levels and significances of serum miR-218 and miR-21 in smokers and COPD patientsObjective To investigate the levels of serum miR-218 and miR-21 in non-smokers without COPD,smokers without COPD,and smokers with COPD,and explore the potential value as biomarkers.Methods Based on Chinese surveillance of COPD,127 volunteers who,including 42 non-smokers without COPD,44 smokers without COPD,and 41 smokers with COPD,did not meet any exclusion criteria and who agreed to take part in the study were selected.The smoking history included age at smoking initiation,years of smoking,number of cigarettes smoked per day,and smoking cessation.The pack-years of smoking were calculated according to the number of packs of cigarettes smoked per day and smoking duration(years).Airflow limitation confirmed by forced expiratory volume 1(FEV1)/forced vital capacity(FVC)value(FEV1/FVC%)<70%.miRNA was extracted from sera,and the expression of miR-218 and miR-21 were determined by q RT-PCR,and its biological function was analyzed.Results1.Effects of CS on pulmonary sfunctionFor the population,the FEV1/FVC% for non-smokers,smokers,and smokers with COPD were 80.8±4.4 %,76.2±4.1%,and 59.7±8.7%,respectively,suggesting that CS induces pulmonary dysfunction.2.Effects of CS on serum miR-218 and miR-21 levelsThe serum miR-218 and miR-21 levels for smokers and smokers with COPD were lower and higher than those of non-smokers,respectively;furthermore,the serum miR-218 and miR-21 levels for smokers with COPD were also lower and higher than those for smokers,respectively.These data suggest that CS reduces miR-218 levels,and increases miR-21 levels in serum.3.The Correlations between the levels of serum miR-218,miR-21 and lung functionCorrelations between the levels of serum miR-218,miR-21 and FEV1/FVC% were analyzed for non-smokers,smokers,and smokers with COPD.The results showed that the levels of serum miR-218 positively correlated with FEV1/FVC%;the levels of serum miR-21 negatively correlated with FEV1/FVC%.These data suggest that the decrease of miR-218 and increase of miR-21 levels in serum are associated with lung dysfunction.Conclusion CS induces the decrease of miR-218 and increase of miR-21 levels in serum.The levels of serum miR-218 positively correlated with FEV1/FVC%,and the levels of serum miR-21 negatively correlated with FEV1/FVC%.In summary,the novel findings of this study are as follows:1.Mi R-218 acts by repressing TNFR1-mediated activation of NF-?B,which is involve in MUC5 AC hyper-production and inflammation in HBE cells induced by cigarette smoke extract.Andrographolide antagonize CSE-induced inflammatory response and mucus hyper-production in HBE cells through induction of miR-218.2.Exosomes derived from HBE cells treated with CS extract causes,in MRC-5 cells,upregulation of miR-21 expression.By targeting p VHL,exosomal miR-21 regulates HIF-1? stabilization,which was associated with myofibroblast differentiation.3.CS induces airway injury,increases of miR-21 levels and decreases of miR-218 levels in lung of mice.Andrographolide prevents CS-induced the downregulation of miR-218 levels,airway inflammation and mucin hyper-secretion in mice.Downregulation of miR-21 prevents CS-induced airway remodeling in mice.4.Cigarette smoking induces aberrant lung function,increases of miR-21 levels,and decreases of miR-218 levels.The levels of serum miR-218 positively correlated with FEV1/FVC%,and the levels of serum miR-21 negatively correlates with FEV1/FVC%. |
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