| BackgroundThe airway inflammatory response caused by harmful particles or gases is the main cause of the pathogenesis and development of Chronic Obstructive Lung Disease(COPD).In recent years,studies have found that traffic related ambient PM2.5(TRAPM2.5)can increase the incidence of COPD,the risk of acute exacerbation,decreased lung function,and increased mortality.Nuclear transcription factor-?B(NF-?B)plays a central controlling and regulating role in the PM2.5 induced inflammatory reaction.NF-?B activation can induce a large number of inflammatory factors,such as IL-6,IL-8,TNF-?,and so on,that can cause chronic and persistent inflammation of the airways and alveoli,and play an important role in the occurrence and development of COPD.Long non-coding RNA(LncRNA)is a type of RNA molecular with a transcript length of more than 200 nt discovered in recent years.They do not encode proteins,but they are expressed in various forms in the form of RNA that is involved in the regulation of gene expression at many levels(epigenetic regulation,transcriptional regulation and post-transcriptional regulation),and the abnormal expression is closely related to the occurrence of respiratory diseases.To elucidate the long chain non-coding RNA genes that play a key role in the pathological process of airway inflammation caused by traffic related ambient PM2.5,and to analyze their relationship with the occurrence and development of chronic obstructive pulmonary disease.It will be possible to provide new biomarkers and drug targets for the diagnosis and treatment of COPD.ObjectiveTo investigate the effects of traffic-related PM2.5 on the inflammatory response of human bronchial epithelial cells and the role and specific molecular mechanisms of long chain non-coding RNAs in order to elucidate the role and mechanism of traffic related PM2.5 in the pathogenesis of chronic obstructive pulmonary disease.Methods and results Part IEffects of traffic related PM2.5 on inflammatory response in human bronchial epithelial cellsMethods:(1)Use a large flow sampler equipped with a PM2.5 cutting head to collect and extract traffic related PM2.5.(2)After stimulating the bronchial epithelial cells of normal humans,the expression of inflammatory cytokines IL-6,IL-8 and TNF-? was detected by ELISA;the protein expression of I???,p-I?B-?,I?B-?,p-p65 and p65 were detected by Western Blot;Fluorescence detected p65 in the cell nucleus.Results:(1)Traffic related PM2.5 was successfully collected and extracted;(2)Expression of IL-6,IL-8,and TNF-? in HBECs stimulated by TRAPM2.5 at 100 ?g/ml increased after 48 h;The protein expression of I???,p-I?B-?,p-p65 and p65 increased;p65 was transferred from cytoplasm to nucleus.Part IIThe role of LncRNA in airway inflammation induced by traffic related PM2.5Methods:(1)Analysis of differential expression profiles of LncRNA and mRNA before and after TRPM2.5 stimulated HBECs using Agilent 4×44K whole-genome microarray;(2)Prediction of LncRNA associated with NF-?B signaling pathway using bioinformatics methods and Q-PCR analysis verification.(3)Q-PCR and ELISA were used to detect the mRNA and protein expression of IL-6,IL-8,TNF-? after interfering with target LncRNA;Q-PCR and Western Blot were used to detect the expression of p65,p-p65,I?B-?,p-I?B-?,and I???,respectively;Fluorescence detected p65 in the cell nucleus.Results:(1)After 48 hours of TRAPM2.5 stimulated to HBECs,microarray analysis showed that 1,698 were up-regulated and 2,321 were down-regulated in differentially expressed LncRNAs;among the differential mRNAs,1,405 were up-regulated and 597 were down-regulated.(2)Three LncRNAs associated with NF-?B signaling pathway were screened out by bioinformatic methods,a LncRNA RP11-86H7.1 was identified and found to be upregulated after TRAPM2.5 stimulated to HBECs cells;(3)After interference with LncRNA RP11-86H7.1,the m RNA levels of p65,I???,I?B-?,IL-6,IL-8 and protein levels of p65,p-p65,IL-6,IL-8 decreased;The number of p65 entering the nucleus decreased.Part IIIThe ceRNA Mechanism of LncRNA RP11-86H7.1 in Traffic associated PM2.5 induced Airway InflammationMethods:(1)The full-length sequence of Lnc RNA RP11-86H7.1 gene was obtained by rapid amplification of cDNA ends(RACE)assay,and analyzed the ability to encode proteins.(2)Fluorescence in situ hybridization(FISH)was used to detect the subcellular localization of LncRNA RP11-86H7.1.(3)The co-expression network of LncRNA RP11-86H7.1—mi RNA—mRNA was constructed using bioinformatic methods.(4)Predictive screening and validation of LncRP11-86H7.1 may serve as a ceRNA targeted miRNA.(5)Q-PCR deteced the oncogene expression of the target miRNA.(6)After mimics transfection of target miRNAs,the mRNA expressions of LncRNA RP11-86H7.1,IL-6,IL-8,TNF-?,p65 and NF?B1 were detected by Q-PCR;the protein expressions of I???,p-I?B-?,I?B-?,p65,p-p65,NF?B1 were detected by WB;IL-6,IL-8,TNF-? levels were detected by ELISA.(7)Dual luciferase reporter assays were performed to detect the interaction targets of LncRNA RP11-86H7.1-miRNA-mRNA and to identify the mi RNA(ceRNA mechanism)shared by LncRNA RP11-86H7.1 and mRNA.(8)The functional recovery experiment: transducted transiently the overexpression of LncRNA RP11-86H7.1 and miR-9-5p mimics or interfered transiently the expression of LncRNA RP11-86H7.1 and miR-9-5p inhibitor were performed to verify the relationship between LncRNARP11-86H7.1—miR-9-5p—NF?B1—the inflammatory phenotype.Results:(1)RACE experiments obtained the full-length sequence of the LncRNA RP11-86H7.1 gene,which does not have the ability to encode proteins.(2)FISH detected the location of LncRNA RP11-86H7.1 in the cytoplasm.(3)We constructed a LncRNA RP11-86H7.1—miRNA—mRNA co-expression network related to NF-?B signaling and inflammatory cytokines expression regulation.(4)Screened and verified that LncRNA RP11-86H7.1 may serve as a targeted silencing miR-9-5p of ceRNA.(5)Q-PCR detected the presence of miR-9-5p in 16 HBE cells.(6)The expressions of p-p65,p-I?B-?,NF?B1,IL-6,IL-8 and TNF-? were significantly down-regulated after transfection of miR-9-5p mimics.(7)Dual luciferase reporter assays confirmed the interaction of LncRNA RP11-86H7.1—miR-9-5p—NF?B1 and confirmed that LncRNA RP11-86H7.1 and NF?B1 have a common binding site point of mi R-9-5p.(8)The functional recovery experiment showed that mi R-9-5p mimics could rescue the levels of NF?B1 and inflammatory factors were up-regulated after overexpressed LncRNA RP11-86H7.1;miR-9-5p inhibitor could rescue the levels of NF?B1 and inflammatory factors were down-regulated after interfered LncRNA RP11-86H7.1.Conclusion:1.Traffic related PM2.5 can induce inflammatory responses in human bronchial epithelial cells.2.Traffic related PM2.5 can up-regulate the expression of LncRNA RP11-86H7.1,activate the NF-?B signaling pathway,and participate in the release of inflammatory factors in human bronchial epithelial cells.3.LncRNA RP11-86H7.1 can adsorb miR-9-5p through competition for endogenous(ceRNA)sponges,thus releasing the inhibitory effect of miR-9-5p on NF?B1,promoting the expression of NF?B1 and inflammatory cytokines,to participate in the airway inflammatory response induced by traffic related PM2.5. |
PDF Full Text Request