Significance And Mechanism Of Mir-424 Expression In Peripheral Blood Of Children With Acute Respiratory Distress Syndrome

Acute lung injury(ALI)/Acute respiratory distress syndrome(ARDS)is one of the critical diseases with high mortality in children.It is a serious lung injury disease caused by various causes.The pathological features are alveolar epithelial and endothelial cell damage and increased alveolar-capillary membrane permeability,resulting in non-cardiogenic acute pulmonary edema.It’s clinical manifestations including severe hypoxia,respiratory distress,gas exchange disturbance,and respiratory mechanical injury.Although the pathogenesis of ARDS is well understood,its regulatory mechanism at the gene level is still unclear.As the molecular mechanism of ARDS is still not fully understood and specific treatment methods are lacking,the mortality rate of ARDS is still up to 30-40%,which is an important cause of death in critically ill children.Although medicine has made great progress recently,the diagnosis and treatment of ARDS remains a challenge.MicroRNA(miRNA,MIR)is a kind of small non coding RNA with a length of about 21～25 bases,which widely exists in eukaryotes.In animals,its mechanism of action is that it binds to the 3 ’non-coding region of target gene in an incomplete complementary way,and plays biological function by inhibiting protein translation.Such regulation generally does not affect the stability of target gene mRNA.miRNAs inhibit the expression of target genes through this mechanism,and thus plays an important role in the synthesis of key proteins,the development of nerve cells,the occurrence of tumors,cell apoptosis and various diseases related to inflammation.miRNA has a wide range of gene regulation functions.The expression of multiple miRNAs in the same cell makes the cell in an internal miRNA environment,which controls thousands of miRNA encoding genes,makes the expression of various proteins at an appropriate level,and regulates the expression of various key proteins.The study of miRNA mechanism may be an important supplement to the study of gene and protein level on the pathogenesis and treatment of ARDS.In the field of ALI/ARDS,the identification of miRNAs associated with the syndrome not only helps us understand the pathophysiological mechanisms involved,but also helps identify potential therapeutic targets.miRNA may be a suitable biomarker for ARDS,and a large number of miRNA have been found to be related to the diagnosis and prognosis of ARDS.With the anticipated advancement of the relationship between miRNAs and ARDS through genome-wide miRNA profiling,more miRNAs related to immune response,inflammatory pathway,disease development and disease severity can be used as a new evaluation tool for diagnosing and monitoring ARDS.Our previous research found that miR-424 was significantly down regulated in the lung tissue of mouse models of ARDS,and target gene prediction and in-depth analysis found that it was related to inflammation,apoptosis,lung surfactant protein,lung development and pulmonary fibrosis.Other studies also found that the expression of miR-424 changed significantly in the alveolar lavage fluid of children with ARDS.miR-424 also affects inflammation,and elevated miR-424 has been found to influence acute stroke by inhibiting lymphocyte and neutrophil inflammation through a CDK6-dependent pathway.However,whether the expression of miR-424 is related to the pathogenesis of human ARDS?What is the specific mechanism of miR-424?Can miR-424 be used as a biological marker for the diagnosis and prognosis of children with ARDS?These are still unclear.This study consists of the following three parts:Part I:To explore the differential expression of miRNA spectrum in peripheral blood mononuclear cells of ARDS children and its possible significance.Part II:Verification of the specific regulatory mechanism of Mir-424 at the cellular level.Part III:Clinical exploration of whether miR-424 can be used as a biological marker for the early diagnosis and prognosis of ARDS children.Part Ⅰ Analysis of miRNAs expression profile in peripheral blood of ARDS childrenObjective:High throughput miRNA sequencing technology was used to screen the expression profile of miRNAs in peripheral blood of children with ARDS,and to screen out the differentially expressed miRNAs and their possible biological functions,so as to provide clues for the study of the pathogenesis of ARDS.Methods:1.Five children with ARDS and five children with sepsis hospitalized in PICU of Henan Provincial People’s Hospital in May 2019 were selected,and five healthy children of the same age who came to the hospital for physical examination during the same period were selected as the control group.2.Early morning fasting peripheral venous blood was collected from each group,and mononuclear cells were isolated.Total RNA was extracted and purified,and miRNA high-throughput sequencing was performed to screen out miRNA with differential expression.3.Target genes of differentially expressed miRNAs were predicted,and GO classification functional annotation analysis and KEGG pathway enrichment analysis of candidate target genes were performed using online database.Results:1.Compared with the control group,there are 33 differentially expressed miRNAs were found in ARDS group,among which 7 miRNAs were up-regulated and 26 miRNAs were down-regulated.2.Compared with the sepsis group,there were 37 differentially expressed miRNAs in ARDS group,of which 4 miRNAs were up-regulated and 33 miRNAs were down-regulated.3.The expression of miR-424 in ARDS group and sepsis group was downregulated compared with the control group,and the expression of miR-424 in ARDS group was also down-regulated compared with the sepsis group.The target genes of target miRNAs were predicted and screened by online database,and the number of candidate target factors was counted.It was found that 132 target genes were predicted by miR-424,and 93 target genes were confirmed.4.Functional annotation analysis of GO classification and KEGG pathway enrichment analysis showed that target genes predicted by differential expression of miRNAs were mainly involved in miRNA regulatory gene silencing,mRNA catabolic process,inflammation related,lung development related,apoptosis related,etc.It is enriched in cell circulation,apoptosis pathway,HIF-1 signaling pathway,MAPK signaling pathway,etc.Conclusions:1.There were a large number of miRNAs with significant differential expression in peripheral blood mononuclear cells of ARDS in children.2.Bioinformatics analysis showed that significantly differentially expressed miRNAs may affect the occurrence and development of ARDS through a variety of biological processes,including immune-related pathways and inflammation-related pathways.3.miR-424 may be involved in the occurrence and development of ARDS in children.Part Ⅱ miR-424 overexpression protects alveolar epithelial cells apoptosis and inflammation by targeting FGF2 through the NF-κB pathwayObjective:To explore the specific mechanism of miR-424 in alveolar epithelial cells through ARDS cell model.Methods:1.The expression of IL-6 and IL-8 in the supernatant of A549 and BEAS-2B cells were determined by ELISA after induction of A549 and BEAS-2B cells with LPS and negative control for 24 h.RT-qPCR was used to determine the relative expression of miR-424 in each group of cells.2.A549 and BEAS-2B cells were transfected with miR-424 mimic,miR-424 inhibitor and their negative control,respectively.The relative expression of miR-424 in each group was determined by RT-PCR after LPS induction.3.A549 and BEAS-2B cells were transfected with miR-424 mimic and miR-424 inhibitor and their negative control,respectively.Apoptosis was detected by TUNEL assay before and after LPS induction.Western Blot was used to detect the expression of cleaved caspase-3 in each group.The expression levels of IL-6 and IL-8 in supernatants of each group were determined by ELISA.The expression of FGF2 protein in each group was detected by Western Blot.4.The possible target genes of miR-424 were predicted using TargetScan and miRDB target gene prediction software,and the targeting relationship between miR-424 and FGF2 was verified by the dual-luciferase reporter gene method.5.A549 and BEAS-2B cells were transfected with adenovirus pAD-FGF2 and FGF2 siRNA and their negative control respectively.After LPS induction,the expression of FGF2 in each group was detected by Western blot.6.A549 and BEAS-2B cells were transfected with FGF2 siRNA,miR-424 inhibitor,FGF2 siRNA+miR-424 inhibitor,adenovirus pAd FGF2,miR-424 mimic,adenovirus pAD-FGF2+miR-424 mimic and negative control.Apoptosis of cells in each group was detected by TUNEL assay after LPS induction.The expression levels of IL-6 and IL-8 in supernatants of each group were determined by ELISA.7.A549 and BEAS-2B cells were transfected with adenovirus pAd FGF2,negative control and blank control,respectively.The expression of nuclear protein p-p65 was detected by Western blot before and after LPS induction.The translocation of p65 was further observed by immunocytochemical staining before and after LPS induction.8.A549 and BEAS-2B cells were transfected with miR-424 inhibitor and its negative control,respectively.Before and after Bayl 1-7082 treatment,the apoptosis of cells in each group was detected by TUNEL assay after LPS induction.The expression levels of IL-6 and IL-8 in cell supernatant of each group were determined by ELISA after LPS induction.Results:1.Compared with the control group,the levels of IL-6 and IL-8 were significantly increased and the expression of mir-424 was significantly down-regulated after lPS-induced alveolar epithelial cells.2.Compared with the control group,the expression of miR-424 in A549 and BEAS-2B cells was increased significantly after transfection with miR-424 mimic,and decreased significantly after transfection with miR-424 inhibitor.3.The apoptosis rate of LPS-induced A549 and BEAS-2B cells was increased significantly after LPS induction.miR-424 mimic significantly reduced the apoptosis of A549 and BEAS-2B cells after LPS induction,and miR-424 inhibitor significantly increased the apoptosis of A549 and BEAS-2B cells after LPS induction.4.Compared with the control group,cleaved caspase-3 was significantly increased in miR-424 inhibitor group and significantly decreased in miR-424 mimic group.5.Compared with the control group,miR-424 mimic inhibited the secretion of IL-6 and IL-8 in LPS-induced cells,while miR-424 inhibitor promoted the secretion of IL-6 and IL-8 in LPS-induced cells.6.According to TargetScan and miRDB target gene prediction software,FGF2 may be one of the target genes of miR-424,and dual luciferase reporter gene detection results also confirmed that FGF2 is the target gene of miR-424.7.Compared with the control group,miR-424 mimic significantly inhibited the expression of FGF2 in LPS-induced cells,and miR-424 inhibitor significantly increased the expression of FGF2 in LPS-induced cells。8.Compared with the control group,the expression of FGF2 protein was significantly increased in the transfected adenovirus pAD-FGF2 cell group,and significantly decreased in the transfected FGF2 siRNA cell group.9.Compared with the control group,silencing FGF2 decreased LPS-induced apoptosis of A549 and BEAS-2B cells,and miR-424 inhibitor increased LPS-induced apoptosis of A549 and BEAS-2B cells.However,miR-424 inhibitor did not regulate the apoptosis of cells with very low FGF2 content in vitro.But,overexpressed FGF2 reversed apoptosis inhibited by miR-424 mimic.10.Compared with the control group,overexpression of FGF2 increased the secretion of IL-6 and IL-8 in LPS-induced A549 and BEAS-2B cells,and miR-424 mimic decreased the secretion of IL-6 and IL-8 in LPS-induced A549 and BEAS-2B cells.Overexpression of FGF2 reversed the secretion of IL-6 and IL-8 in LPS-induced A549 and BEAS-2B cells inhibited by miR-424 mimic.Silencing FGF2 reduced the secretion of IL-6 and IL-8 in LPS-induced A549 and BEAS-2B cells with or without miR-424 inhibitor.11.Compared with the control group,LPS-induced nuclear protein p-p65 expression level of A549 and BEAS-2B was increased,and overexpression of FGF2 increased LPS-induced nuclear protein p-p65 expression level of A549 and BEAS-2B.Before LPS induction,p65 was mainly expressed in cytoplasm.p65 gradually entered the nucleus 24 h after LPS induction.Transfection of adenovirus pAd FGF2 further promoted the expression of p65 in the nucleus.12.Compared with the control group,BAY11-7082 treated cells attenuated apoptosis and IL-6 and IL-8 secretion caused by miR-424 inhibitor.Conclusions:1.miR-424 regulates LPS-induced apoptosis and inflammatory response of alveolar epithelial cells by targeting FGF2.2.miR-424 regulates apoptosis and inflammation of alveolar epithelial cells through NF-κB pathwayPart Ⅲ The prognostic value of miR-424 in evaluating the diagnosis and prognosis of ARDS childrenObjcctive:To investigate the predictive value of miR-424 on the risk of sepsis complicated with ARDS and its correlation with the prognosis of ARDS children.Methods:1.In this paper,a prospective study was conducted to collect general data of children with sepsis(including ARDS)admitted to our hospital from February 2020 to February 2021.According to whether sepsis is complicated with ARDS,they were divided into sepsis complicated with ARDS group and sepsis complicated without ARDS group.At the same time,20 healthy children of the same age were selected as the control group.ARDS occurrence and ARDS mortality records were recorded in all children with sepsis,and their blood samples were collected.The expression of miR-424 in peripheral blood mononuclear cells was detected by RT-qPCR.2.Pearson correlation analysis was used to explore the relationship between the expression of miR-424 and Pediatric Critical Illness Score(PCIS),Sequential organ failure assessment(SOFA),P/F value(PaO2/FiO2),C-reactiveprotein(CRP),procalcitonin(PCT),IL-6 and IL-8 in children with sepsis complicated with ARDS.3.Area under the Receiver operating characteristic curve(AU-ROC)was used to evaluate the predictive value of miR-424 on the risk of sepsis complicated with ARDS.4.Children with sepsis complicated with ARDS were divided into survival group and death group,and the expressions of miR-424,IL-6 and IL-8 in survival group and death group were detected.The factors affecting the prognosis of the children with ARDS were analyzed by multivariate Logistic regression.Area under the Receiver operating characteristic curve(AU-ROC)was used to evaluate the risk of death of miR-424 in patients with sepsis complicated with ARDS.Results:1.A total of 121 sepsis patients were included in this study,including 36 sepsis patients with ARDS.The expression level of miR-424 in the blood of patients with sepsis complicated with ARDS was significantly lower than that of patients with sepsis complicated without ARDS.2.In sepsis complicated with ARDS group,the expression of miR-424 was negative correlated with the levels of IL-6,IL-8 and CRP.The expression of miR-424 was positive correlated with PCIS score,P/F value and albumin.3.Receiver operating characteristic curve(ROC)showed that miR-424(AUC:0.908,95%Cl:0.856-0.960)could differentiate patients with sepsis complicated with ARDS from those without ARDS.4.Compared with survival group,miR-424 was significantly decreased in death group.Multivariate Logistic regression analysis showed that the expression of miR-424 and albumin were independent risk factors for death in ARDS children.5.The decrease of miR-424(AUC:0.845,95%CI:0.696-0.995)predicted an increased risk of death in children with ARDS.Conclusions:1.miR-424 has predictive value for the early diagnosis of ARDS in children.2.miR-424 has predictive value for the prognosis of ARDS in children.