Expression And Bioinformatics Analysis Of MiRNA In Serum Exosomes In COPD Rats

Objective:Mi RNA derived from exosomes can regulate important cell activities and participate in the occurrence and development of COPD.The purpose of this study is to investigate the expression of mi RNA in serum exosomes in COPD rats,and to clarify the role of mi RNA in serum exosomes in COPD by bioinformatics analysis and experimental verification.Methods:1.Establishment of COPD animal model: Twelve male SD rats aged 6-8 weeks and weighing 160-180 g were randomly divided into experimental group and control group.COPD rat model was established by passive fumigation and intratracheal instillation of porcine pancreatic elastase.After 15 weeks of passive smoking,the pulmonary function examination was completed,and the rats were killed by cervical dislocation.Lung tissue was isolated and serum was collected.Serum was used to extract exosomes,and lung tissue was embedded into wax blocks for HE staining.2.Serum exosomes isolation and mi RNA extraction: The exosomes were separated by ultracentrifugation,the particle size of exosomes was observed and analyzed by electron microscope,and the exosome marker protein was detected by Western blot.The extracted exosomes use RNA lysate to extract mi RNA in exosomes,and detect the concentration and purity of mi RNA.3.Bioinformatics analysis of mi RNA in exosomes: After normalizing the initial data with Illumina of R software,use | log2(FC)| ≥ 0.5849;P value ≤ 0.05 was used as the screening standard to screen the differentially expressed genes related to COPD,predict the target genes of the differentially expressed mi RNAs,and analyze the go enrichment and KEGG signal pathway of the target genes.4.Cell experiment verification: After preparing cigarette smoke extract,human bronchial epithelial cells were treated with 0.5%,1% and 2% cigarette smoke extract respectively,and the cell invasion ability was detected by Transwell method.The appropriate concentration of cigarette smoke extract was selected,and the model of three-dimensional external intervention in human bronchial epithelial cells was established.The expression level of mi RNA was detected by q PCR and detected by q PCR and Western blot α-SMA、 α-catenin,E-cadherin and N-cadherin.Results:1.COPD modeling(1)General manifestations: The rats in the control group had stable breathing,normal skin and hair luster,normal weight gain and normal defecation.The rats in the experimental group had shortness of breath,some of them breathed with their mouths open,the skin and hair lacked luster,and some of them had depilation.They sweated more during smoking,ate less water than the control group,lost weight,and had dry stools.(2)Lung function: Compared with the control group,FVC、FEV0.3、FEV0.3/FVC、PEF decreased significantly(P < 0.05).(3)HE staining of lung tissue: In the control group,the small airway wall was normal,without obvious fracture and thickening,no inflammatory cell infiltration,and the alveolar structure was normal.In the experimental group,the bronchial wall of the small airway was thickened,the inflammatory cells around the airway were infiltrated,the alveolar structure was disordered,showing cystic expansion,and some alveoli fused into pulmonary bullae.2.Serum exosomes and mi RNA: Electron microscopic quality evaluation of exosomes: complete in shape,spherical and uniform in size;Quality evaluation of exosome particle size: The particle concentration is 10 ^ 7 ～ 10 ^ 11 particles / ml,and the particle size is 30 – 140nm;Quality evaluation of exosome protein: CD9,CD63 and TSG101 have bands;Total RNA extracted: 45.0-79.1ng.3.Bioinformatics analysis of secrete mi RNA: The differentially expressed mi RNAs in serum secretes were analyzed by bioinformatics method,and 15 differentially expressed mi RNAs were screened,including 8 up-regulated mi RNAs and7 down-regulated mi RNAs.4.Cell experiment verification :After treating human bronchial epithelial cells with different concentrations of cigarette smoke extract,0.5% concentration interferes with the enhancement of cell invasiveness.This concentration is selected for the next experiment.After the cells were treated with 0.5% cigarette smoke extract,the expression of mi R-182-5p and mi R-185-5p decreased by q PCR(P < 0.05).After the same concentration of cigarette smoke extract interferes with the cells,The expression of α-catenin and E-cadherin decreased.The expressions of α-SMA and N-cadherin were significantly increased(P < 0.05).Conclusion:1.Screening the differentially expressed mi RNAs in COPD rat model,a total of15 differentially expressed mi RNAs were obtained,including 8 up-regulated mi RNAs and 7 down-regulated mi RNAs.2.The expression of mi R-182-5p and mi R-185-5p from the selected exosomes decreased,suggesting that they may be involved in the occurrence and development of COPD.3.Differentially expressed mi RNAs may participate in the occurrence and development of COPD through epithelial mesenchymal transition of bronchial epithelial cells.