The Role Of Mir-337-3p In The Pathogenesis Of OSA By Regulating The Expression Of TLR4

Obstructive sleep apnea(OSA)is a common sleep-disordered breathing disorder characterized by repeated episodes of complete or partial collapse of the upper airway during sleep,with apnea and/or hypopnea resulting in hypoxemia,microarousal,hyperventilation,and normal blood oxygenation.Such changes in blood oxygen occur in cycles during sleep at night,that is,repeated hypoxia/reoxygenation.The process is similar to ischemia/reperfusion injury,which produces a large number of reactive oxygen species and causes oxidative stress response in the body,and then leads to cell damage,local and systemic inflammatory response,and the production of a variety of inflammatory factors,such as tumor necrosis factor-α(TNF-α),C-reactive protein(CRP),endothelin-1(ET-1)and other increased release,activation of inflammation"waterfall effect",further aggravating cell and tissue damage,leading to multiple organ system dysfunction.OSA is the source of a variety of chronic diseases,seriously harm to human health.The pathogenesis of OSA is still unclear.Current studies mainly focus on intermittent hypoxia and sleep fragmentation,and intermittent hypoxia is more studied.MicroRNAs(miRNAs)have been reported to play an important regulatory role in the occurrence and development of a variety of diseases.MiRNAs are small non-coding RNAs with a length of 20 to 22 nucleotides.By binding to the 3’UTR of target mRNAs,miRNAs play a role in negatively regulating the expression of target genes at the post-transcriptional level.The expression of miRNAs is closely related to lung growth and development,maintenance of lung homeostasis,cause lung inflammation.However,the molecular mechanism of miRNAs in OSA-induced lung injury remains unclear.In this study,by establishing rat models of intermittent hypoxia at different concentrations,miRNA microarray detection was performed on rat lung tissues,and specific miRNAs in the lung tissues of intermittent hypoxia rats were screened out to establish miRNA expression profile.MiR-337-3p was selected for validation in rat lung tissue,and TLR4 was predicted and verified as the target gene of miR-337-3p.In vitro experiments were conducted to investigate the effects of up-regulation/down-regulation of miR-337-3p and down-regulation of TLR4 on proliferation and apoptosis of intermittent hypoxia alveolar epithelial cells and its downstream molecular regulation mechanism.In vivo experiments were conducted to explore the alleviating effects of miR-337-3p mimic and siTLR4 on lung tissue injury in intermittent hypoxia rats.Through the above experiments,the molecular mechanism of miR-337-3p in OSA-induced lung injury was investigated.This study mainly consists of the following three parts:Part 1 Construction of rat model of chronic intermittent hypoxia and analysis of miRNA expression profile[Objective]The rat model of intermittent hypoxia(IH)at different concentrations was established to observe the damage of the lung tissue of rats,and the differential expression profile of miRNAs in the lung tissue of rats with intermittent hypoxia(IH)at different concentrations were analyzed.[Methods]Thirty adult male Wistar rats(weight:200～220 g)were used.All rats were randomly divided into 5 groups,namely 5%oxygen concentration group(IH1),7.5%oxygen concentration group(IH2),10%oxygen concentration group(IH3),12.5%oxygen concentration group(IH4)and blank control group(NC).Rats were exposed to intermittent hypoxia for 8 hours a day for 12 weeks.HE staining and TUNEL staining were used to observe the lung tissues of rats.MiRNA microarray was used to analyze miRNAs expression profile in lung tissues of rats in each group.[Results]HE staining was used to observe the lung tissue of rats.Except for the control group,the lung tissue of rats in the intermittent hypoxia group was damaged to varying degrees,which was manifested as alveolar structure destruction,inflammatory cell infiltration and lung interstitial thickening,and the more severe the hypoxia,the more obvious the lung tissue damage was.TUNEL staining showed that with the decrease of oxygen concentration,the proportion of round cells and brown-yellow nuclei in lung tissue cells increased,indicating the increase of apoptosis.MiRNAs chip analysis results showed that there were a large number of differentially expressed miRNAs in the lung tissues of the four groups of rats.The intersection of miRNAs expression profiles of groups IH1-4 was taken to obtain 10 different miRNAs shared by groups IH1-4,which were:hsa-miR-122-5p,hsa-miR-30 c-2-3p,hsa-miR-363-3p,hsa-miR-199a-3p,hsa-miR-127-3p,hsa-miR-33a-5p,hsa-miR-205-5p,mmu-miR-376c-3p,hsa-miR-369-5p,rno-miR-337-3p.According to literature review,6 of 10 common miRNAs have been reported in ischemic hypoxia diseases,including miR-337-3p.Cluster analysis showed that miR-337-3p was consistently down-regulated in normal control group and intermittent hypoxia group.We focused on the role of miR-337-3p in OSA.The 10 miRNAs common seed region base sequences were highly conserved among different species.Target gene prediction,GO analysis and pathway prediction of miRNAs in IH1～4 groups were performed by Funrich software.The results showed that the 10 miRNAs contained 929 target genes,and the cell components were mainly distributed in the nucleus,followed by the cytoplasm.The main molecular function is the activation of transcription factors.Biological processes focus on cell interaction and signal transduction.Pathway analysis included many target gene signaling pathways,including LKB1 signaling pathway,Nectin adhesion pathway and S1P pathway.[Conclusion]Intermittent hypoxia can cause lung tissue damage,the more serious hypoxia damage is more obvious.Intermittent hypoxia can cause apoptosis of lung tissue cells,and the more serious hypoxia,the higher the percentage of apoptosis.Different miRNAs were found in lung tissues of rats with intermittent hypoxia at different concentrations.There were 10 miRNAs in lung tissues of rats with intermittent hypoxia at different concentrations,and miR-337-3p was selected for further study.MiRNAs may play a role in OSA through LKB1,Nectin and S1P pathways.Part 2 Expression and target gene verification of miR-337-3p in intermittent hypoxia rats[Objective]To verify the expression of miR-337-3p in intermittent hypoxia rat lung tissues,predict target genes,and verify the interaction between miR-337-3p and target genes.[Methods]MiR-337-3p was selected for further study,and its expression was verified by qPCR experiment.TargetScan predicted the target gene of miR-337-3p online,and double luciferase reporter gene experiment was used to verify whether miR-337-3p could target the target gene.QPCR and ELISA were used to detect the expression levels of target genes in lung tissues of rats in each group.Western blot was used to detect the expression of target protein in lung tissues of rats in each group.Immunohistochemistry was used to detect the localization and expression of target protein in rat lung tissue cells,and ELISA was used to detect the expression level of inflammatory factors in lung tissue.[Results]MiR-337-3p was selected for qPCR verification in intermittent hypoxia rat model.The results showed that compared with NC group,the expression of miR-337-3p in lung tissues of rats in IH1-IH4 groups was significantly down-regulated,and the lower the oxygen concentration was,the more obvious the down-regulation was,and the miR-337-3p in rats in IH1 group was the most significantly down-regulated.TargetScan online prediction showed that there were complementary binding sites between miR-337-3p and TLR4 mRNA.Double luciferase reporter gene assay confirmed that miR-337-3p could directly bind TLR4 mRNA,and miR-337-3p was negatively correlated with TLR4 mRNA.This indicates that miR-337-3p targets TLR4.QPCR and ELISA showed that the expression level of TLR4 in lung tissues of intermittent hypoxia rats was up-regulated,and the more severe hypoxia,the higher the expression level of TLR4.Inaddition,IHC staining showed positive expression of TLR4 in the lung tissues of chronic intermittent hypoxia rats.The more severe hypoxia,the more obvious expression of TLR4.The expressions of inflammatory factors TNF-α,IL-1β and IL-6 in the lung tissues of IH1-IH4 group were significantly increased by ELISA,and increased with the severity of hypoxia.[Conclusion]MiR-337-3p was down-regulated in intermittent hypoxia rats,and the more severe hypoxia,the more down-regulated miR-337-3p was.TLR4 is the target gene of miR-337-3p,which is highly expressed in the lung tissues of intermittent hypoxia rats and increases with the severity of hypoxia.The expression levels of TLR4 and miR-337-3p in lung tissues of intermittent hypoxia rats were significantly negatively correlated.The expressions of inflammatory factors TNF-α,IL-1β and IL-6 in lung tissues of intermittent hypoxia rats were increased.Part 3 The regulatory relationship and mechanism of miR-337-3p and TLR4[Objective]The regulatory relationship between miR-337-3p and TLR4 was investigated through intermittent hypoxia cell model and rat model,and the molecular mechanism of lung injury caused by miR-337-3p in intermittent hypoxia was elucidated.[Methods]The intermittent hypoxia cell model was constructed,and the cells were transfected with miR-337-3p mimic,miR-337-3p inhibitor and TLR4 siRNA.The expression levels of miR-337-3p and TLR4 were detected by qPCR.CCK-8 method was used to detect the proliferation of alveolar epithelial cells in each treatment group,and flow cytometry was used to detect the apoptosis of alveolar epithelial cells in each treatment group.Western blot assay was performed to detect the expression levels of MyD88,IRAK1,IRAK4,TRAM,TRAF6,NF-κB P65 and p38MAPK in TLR4 signaling pathway,and to observe the response of pathway agonists and inhibitors to the effects of pathway.MiR-337-3p mimic and TLR4 siRNA were injected into the tail vein of intermittent hypoxia rat models,and the mRNA expression levels of miR-337-3p and TLR4 in lung tissues were detected by qPCR.Western blot was used to detect TLR4 expression in lung tissues of rats.[Results]Overexpression of miR-337-3p and knockdown of TLR4 can promote the proliferation of intermittent hypoxia alveolar epithelial cells,and reduce the apoptosis of intermittent hypoxia alveolar epithelial cells.The increase of miR-337-3p can inhibit the expression of TLR4,and the decrease of miR-337-3p can increase the expression of TLR4,and the two are negatively regulated.Overexpression of miR-337-3p inhibited the activation of NF-κB pathway,thereby reducing the expression of TLR4,MyD88,IRAKI,IRAK4,TRAM,TRAF6,NF-κB p65,p38MAPK proteins and inflammatory factors TNF-α,IL-6,IL-1β.After the injection of miR-337-3p mimic,the expression of miR-337-3p was increased and TLR4 was decreased in the lung tissues of rats.After injection of siTLR4,TLR4 expression in rat lung tissue was decreased.Apoptosis and tissue damage were improved in miR-337-3p mimic injection group and TLR4 siRNA injection group.[Conclusion]MiR-337-3p had a negative regulatory relationship with TLR4.Up-regulation of miR-337-3p and down-regulation of TLR4 promoted cell proliferation,inhibited apoptosis and improved tissue damage.MiR-337-3p mediated OSA-induced lung injury by targeting TLR4/NF-κB/MAPK signaling pathway.