A Study On The Molecular Mechanisms Of MiR-199a-3p Underlying The Pathogenesis Of Acute Respiratory Distress Syndrome Caused By Sepsis

Objective Acute respiratory distress syndrome(ARDS)is a common clinical critical disorder of the respiratory system,and sepsis is the most important cause of ARDS.Inflammation plays an important role in regulating the pathogenesis of ARDS,but its specific molecular signal regulation pathway is still not fully understood.In recent years,microRNAs have been found to play an important regulatory role in the pathogenesis of many diseases,but it is still unclear which microRNAs are involved in the pathogenesis of ARDS.Based on these observastions,the study intended to determine the microRNA expression profile in the lung biopsies from the patients with ARDS and non-ARDS and then identify the differentially expressed microRNAs.We would predict the potential target genes of the aberrantly expressed microRNAs,explore whether these microRNAs can target genes that are involved in inflammation signaling pathways.We would also investigate how microRNAs regulate the expression of their target genes and affecting the signaling pathways in which the target genes participate.Meanwhile,we planned to explore the molecular mechanism of microRNA aberrant expression in order to describe a complete signal pathway for ARDS pathogenesis,which may provide more potential targets for inhibiting ARDS inflammation and provide more options for the therapy of ARDS.Methods We collected lung tissue samples from patients with sepsis-induced ARDS and non-inflammatory controls and obtained differentially expressed microRNAs through microRNA microarray analysis.Treating one human epithelial cell A549,one human macrophage U937 and one microglia cell line HMC3 with endotoxin(LPS)to activate the inflammatory signal pathway,we detected the expression of those differentially expressed microRNAs found in microarray in each cell line to determine if the inflammatory microenvironment could affect their expression.One database(www.mirdb.org)was used to predict the target genes of microRNAs affected by the inflammatory microenvironment.At the same time,we performed a gene-level microarray analysis in the same ARDS and control samples to obtain differentially expressed genes,and then compared the microRNA target genes predicted by the database with the differentially expressed genes found in the microarray and found the overlapped genes for research.Different strategies,including cell transfection,ELISA and Western blotting,were used to detect whether overexpression or antagonistic microRNA expression affected the expression of its target genes and downstream molecules,which would help to confirm the relationship between the microRNAs and their target genes.Finally,we also clarified the molecular mechanism underlying microRNA aberrant expression by analyzing their promoters to determine whether they were regulated by DNA methylation or transcription complex.We analyzed the possible transcription factor binding sites in their promoter regions in the database(www.alggen.lsi.upc.es),and also knocked down or overexpress each transcription factor in the human cell line to observe their effects on microRNA expression in order to identify the transcription factor that regulated the expression of the microRNAs.Next,we performed immunoprecipitation and mass spectrometry to identify possible members of the transcription complex bound by the transcription factor.By knockdown or overexpression of each member of the transcription complex,we detected their effects on microRNAs and the target genes of microRNAs.Results miR-199a-3p was significantly downregulated in ARDS-biopsies through microarray technology.In vitro studies revealed that the expression of miR-199a-3p in LPS-treated cell lines was also inhibited.Mechanically,miR-199a-3p specifically bound to 3’-UTR of NLRP1,one of the key members of the NLRP1 inflammasome.Ectopic overexpression or downregulation of miR-199a-3p resulted in the repression or induction of NLRP1,respectively,thereby downregulating or activating its downstream events.Through prediction,we found that the upstream region(-2000 bp)of miR-199a-3p had potential binding sites for the transcription factors TCF4,FOXP3,AP1,HIF1,and NF-κB.We individually knocked down or overexpressed these transcription factors and found only the changes of FOXP3 could affect the expression of miR-199a-3p.Using immunoprecipitation(IP),mass spectrometry and co-IP assays,we found that FOXP3 formed a transcriptional complex with HDAC1(histone deacetylase 1)and CtBP2(C-terminal-binding protein 2).The CtBP2-HDAC1-FOXP3 transcriptional complex(CHFTC)could specifically bind to the promoter of miR-199a-3p and regulate its expression.Conclusions : CtBP2 acts as a transcription repressor and forms a transcription complex with HDAC1 and FOXP3,inhibiting the expression of miR-199a-3p,and finally up-regulating the NLRP1 gene.The activated NLRP1 protein assembles into inflammasomes to activate capase-1,which cleaves the precursor of interleukin-1β(pro-IL-1β)and the precursor of interleukin-18(pro-IL-18),thereby processing the maturation and secretion of IL-1β and IL-18.IL-1β further activates the IL-1 receptor complex and induces the expression and activation of a variety of cytokines involved in inflammatory cascades.Increased inflammation Immune response promotes the permeability of alveolar epithelial cells,aggravates pulmonary edema and lung damage,eventually leading to the pathogenesis of ARDS.