Study Of Changes And Clinical Significance Of Serum Exosomes MicroRNAs Profiles In Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic interstitial pulmonary fibrosis（IPF）is a lung disease of unknown origin that occurs in middle-aged and elderly men,with chronic,progressive interstitial fibrotic lesions as the main pathological manifestation.Chronic inflammatory lesions in the alveoli,interstitium,small blood vessels,and terminal airways,as well as fibrotic lesions that develop during the course of prolonged inflammation,can be observed in IPF.The triggering and persistent pathogenic mechanism of IPF is still unclear,and the currently available drugs can only delay the decline of lung function in patients,and there is no effective treatment so far except for lung transplantation.Exploring the pathogenesis of IPF will provide possible targets for its early clinical intervention and has important clinical value for the diagnosis and treatment of IPF.Exosomes are a class of doublelayered phospholipid enveloped vesicles containing proteins,lipids,DNA,RNA and other substances,which are widely found in many body fluids.In recent years,exosomes have been called "messengers" for intercellular information transfer,and they are involved in the occurrence and development of many diseases through the genetic material they carry,and exosomal micro RNAs have received more attention.The expression profile of exosomal micro RNAs in serum of IPF patients is still unclear.ObjectiveTo investigate the changes in the expression profile of serum exosomal micro RNAs in patients with IPF and the possible clinical significance of serum exosomal micro RNAs in IPF.MethodsTwenty-four patients with primary IPF who attended the Department of Respiratory Medicine at the First Affiliated Hospital of Xinxiang Medical University from January2019 to January 2021 were selected as the IPF group,and another 24 patients matched for age and gender with the IPF group and who underwent a health checkup at our hospital were selected as the normal control group（NC group）,for a total of 48 subjects to be included in the study.Baseline clinical information was collected from enrolled patients:age,sex,smoking history,and pulmonary function test results: total lung capacity（TLC）,forced expiratory volume in one second / predict value,（FEV1% pred）,carbon monoxide diffusing capacity /predict value（DLCO%pred）.Fasting blood was collected from all subjects,and serum exosomes were separated from both groups by low-temperature high-speed centrifugation.The extracted exosomes were identified morphologically by transmission electron microscopy.Total exosomal RNA was extracted,and after quality control,the changes in the expression profiles of exosomal micro RNAs in both groups were detected by high-throughput sequencing technology.By bioinformatics method,target gene prediction and functional analysis of differentially expressed micro RNAs were performed to explore the abnormal signaling pathways involved in differentially expressed micro RNAs,correlate the expression of differentially expressed micro RNAs with the trend of lung function changes in IPF patients,and screen the possible mechanisms and targets of the abnormal expression profiles of serum exosomal micro RNAs involved in the occurrence of IPF To screen the possible mechanisms and targets of the abnormal expression profiles of serum exosomal micro RNAs involved in IPF.Results1.Comparison of baseline data between the two groups: differences in age（57.83±6.72 vs 59.79±5.99 years）,gender ratio（41.7% vs 54.2%）for males,and smoking history（25% vs 37.%）were not statistically significant（P >0.05）between the two groups.TLC,FEV1% pred,DLCO%pred in the IPF group were lower than in the NC group（P <0.01）。2.Identification of serum exosome: the two groups of isolated extracted serum exosomes were identified by transmission electron microscopy.A biconcave circular vesicle structure with a diameter of about 100 nm could be seen under the microscope,identifying the extracted components as exosomes.3.Exosomal micro RNA high-throughput sequencing: the number,types and expression level of exosomal mi RNAs were found to be different between the two groups.A total of 168 micro RNAs were differentially expressed between the two groups.Compared with the NC group,64 micro RNAs were up-regulated and 104 micro RNAs were down-regulated in the IPF group.Among the top 5 micro RNAs with the highest abundance in both groups,a total of 3 micro RNAs were involved in PI3K/Akt/m TOR signaling pathway,among which mi R-1-3p and mi R-99a-5p were more expressed in the NC group than in the IPF group,and the differences were statistically significant（P <0.05）,and the expression of mi R-1b in the NC group was higher than that in the IPF group,but the difference was not statistically significant（P > 0.05）.Sorting the micro RNAs between the two groups in order of difference significance from highest to lowest,it was seen that the micro RNA with the most significant difference between the two groups was mi R-672-5p（P < 0.05）,which was involved in the TNF signaling pathway,and mi R-411-5p,which was involved in the m TOR signaling pathway,was differentially expressed between the two groups（P < 0.05）.4.3 micro RNAs involved in PI3 K / Akt / m TOR signaling pathway and differentially expressed between the two groups: mi RNAs mi R-1-3p,mi R-99a-5p,mi R-411-5p were positively correlated with DLCO%pred in IPF patients（P < 0.05）.The correlation coefficient r between mi R-99a-5p and DLCO%pred was 0.6172,the correlation coefficient r between mi R-1-3p and DLCO%pred was 0.5748 and the correlation coefficient r between mi R-411-5p and DLCO%pred in IPF patients was 0.825.ConclusionSerum exosomal micro RNA expression profiles were altered in patients with IPF compared with the normal subjects,and abnormally expressed exosomal micro RNAs may be involved in IPF progression through activation of the PI3K/Akt/m TOR signaling pathway.