MiR-9 Targeting RUNX1 Regulation And Mechanism Of Alveolar Procoagulation And Fibrinolysis Inhibition In ARDS

Objective:Massive intra-alveolar fibrin deposition is one of the key causes of intractable hypoxemia and high morbidity and mortality in acute respiratory distress syndrome(ARDS).Enhanced intra-alveolar procoagulation and inhibition of fibrinolysis are key to cause fibrin deposition,the nuclear factor kappa-B(NF-κB)signalling pathway has a regulatory role in alveolar procoagulation and fibrinolysis inhibition,but the regulatory mechanism is unclear.It was shown that miR-9(microRNA-9a-5p),an important regulator of ARDS,interacts with the NF-κB signalling pathway,and Runtrelated transcription factor 1(RUNX1)is highly expressed in alveolar epithelial cells,and RUNX1 has been confirmed as a target gene of miR-9.This paper aimed to investigate the regulatory effects of miR-9 targeting RUNX1 on alveolar procoagulation and fibrinolysis inhibition in ARDS and to explore the regulatory mechanisms to provide a new basis for the pathogenesis of ARDS and the search for effective therapeutic targets.Methods:1.miR-9/RUNX1 expression in ARDS patients Patients with ARDS who were admitted to the Intensive Care Unit(ICU)for the first time and non-ARDS patients who were admitted at the same time were included in the study.The expression of miR-9 and RUNX1 m RNA in peripheral venous blood of each group of patients was measured by quantitative real-time PCR(qPCR),and clinical data was collected to observe the expression levels of miR-9 and RUNX1 in ARDS disease and the correlation between them.2.miR-9 ameliorates alveolar procoagulation and fibrinolytic inhibition in ARDS rats(1)To construct a Lipopolysaccharides(LPS)-induced ARDS model in rats,Hematoxylin-eosin staining(HE)was used to detect histopathological changes in the lungs and to clarify the successful replication of the ARDS rat model.(2)After tail vein injection of miR-9 agonist(miR-9 agomir),miR-9 agonist negative control(miR-9 agomir-NC),miR-9 inhibitor(miR-9 antagomir)and miR-9 inhibitor negative control(miR-9 antagomir-NC)for 48 h,lipopolysaccharide was inhaled.Lipopolysaccharide(LPS)(10 mg/kg)was inhaled to replicate the ARDS model.The experimental groups were as follows: normal group(Control group),LPS group,LPS+ miR-9 agomir group,LPS+ miR-9-NC group,LPS+ miR-9 antagomir group,LPS+ miR-9 antagomir-NC group.(3)miR-9 expression in rat lung tissues: qPCR to detect miR-9 expression in lung tissues:(4)Effect of miR-9 on lung injury in ARDS rats: detection of alterations in lung histopathology in ARDS rats using HE staining;detection of lung injury coefficient,alterations in lung wet/dry weight ratio.(5)Effect of miR-9 on alveolar procoagulation and fibrinolysis inhibition in ARDS rats: qPCR and WB techniques were applied to detect the changes of m RNA and protein related to Tissue factor(TF),Plasminogen activator inhibitor-1(PAI-1).Alveolar lavage fluid(Bronchoalveolar Lavage Fluid,BALF)was collected to detect TF,PAI-1,and thrombin activity.The expression of collagen III(Collagen III)in lung tissue was detected by immunohistochemistry.(6)Effect of miR-9 on NF-κB signalling pathway: WB was applied to detect the changes of NF-κB signalling pathway related proteins.(7)Effect of miR-9 on RUNX1 gene expression: qPCR and WB techniques were used to detect the changes in RUNX1 m RNA and protein.3.Mechanisms of miR-9 effects on alveolar procoagulation and fibrinolysis inhibition(1)Rat alveolar type II epithelial cell(RLE-6TN)was cultured in vitro,and RLE-6TN cells were stimulated with LPS.(2)By constructing lentiviral(LV)overexpression of miR-9 gene(LV-miR-9)and its negative control lentiviral vector(LV-control),low expression(LV-miR-9-inhibitor)and its negative control lentiviral vector(LV-control),RUNX1 gene overexpression(LV-RUNX1)and its negative control lentiviral vector(LV-control)and transfected RLE-6TN cells,respectively.In addition,the overexpression miR-9 group(LV-miR-9)+ overexpression RUNX1 group(LV-RUNX1)were constructed and cultured for stable expression of cell lines.(3)Effect of miR-9 on alveolar procoagulation and fibrinolysis inhibition: The expression of TF and PAI-1 m RNAs and proteins in RLE-6TN cells after transfection with LV-miR-9 and LV-miR-9-inhibitor were clarified by qPCR and WB techniques.(4)Effect of miR-9 on NF-κB signaling pathway: The changes of NF-κB signaling pathway related proteins after transfection of RLE-6TN cells with LV-miR-9 and LVmiR-9-inhibitor were clarified by WB technique.(5)Targeted regulation of RUNX1 by miR-9: The expression of RUNX1 m RNAs and proteins after transfection of RLE-6TN cells with LV-miR-9 and LV-miR-9-inhibitor was clarified by qPCR and WB techniques,and the direct targeting of miR-9 to RUNX1 was confirmed by dual luciferase reporter gene assay.(6)Effect of RUNX1 on NF-κB signaling pathway: WB assay was applied to clarify the changes of NF-κB signaling pathway related proteins after transfection of RLE-6TN cells with LV-RUNX1.CO-IP was applied to verify the interaction of IKKβwith RUNX1 protein.(7)miR-9 targets RUNX1 to regulate alveolar procoagulation and fibrinolysis inhibition: construct an overexpression miR-9(LV-miR-9)+ overexpression RUNX1group(LV-RUNX1)cell line and verify the effect of miR-9 on NF-κB signaling pathway and alveolar procoagulation and fibrinolysis inhibition through RUNX1 by qPCR and WB technique using Rescue assay.effects.Results:1.miR-9/RUNX1 expression in ARDS patients qPCR assay showed that miR-9 levels in the peripheral blood of ARDS patients were lower than those in controls.In contrast,the m RNA expression of RUNX1 was significantly increased in ARDS patients;the expression levels between RUNX1 and miR-9 showed a significant negative correlation in ARDS patients.2.miR-9 ameliorated alveolar procoagulation and fibrinolysis inhibition in ARDS rats(1)The LPS-induced ARDS model was successful: HE staining showed: massive inflammatory cell infiltration in lung tissue,increased alveolar destruction and alveolar exudation,etc.The Diffuse Alveolar Damage(DAD)score was significantly increased.(2)Expression of miR-9 in rats of all groups: compared with the Control group,miR-9 expression in lung tissues of ARDS rats was significantly down-regulated.miR-9expression in the miR-9 antagomir group was more significantly reduced,while miR-9 levels in lung tissues of rats in the miR-9 agomir group were significantly higher than those in ARDS rats.(3)Effect of miR-9 on lung injury in ARDS rats: HE staining showed that miR-9agomir significantly improved lung injury pathological score and decreased lung wet/dry weight ratio in ARDS rats,while miR-9 antagomir aggravated lung injury pathological score and lung wet/dry weight ratio in ARDS rats.(4)Effects of miR-9 on alveolar procoagulation and fibrinolysis inhibition in ARDS rats: qPCR and WB results showed that miR-9 agomir significantly decreased the m RNAs and protein expression levels of TF and PAI-1 under LPS induction;miR-9antagomir significantly increased the m RNAs and protein levels of TF and PAI-1under LPS induction.ELISA results showed that miR-9 agomir significantly decreased the activity of TF,PAI-1 and thrombin in BALF under LPS induction,and miR-9 antagomir promoted the expression of the above indicators.Immunohistochemical results showed that miR-9 agomir decreased Collagen III expression in lung tissues,while miR-9 antagomir significantly increased Collagen III content in lung tissues.(5)Effect of miR-9 on NF-κB signalling pathway: WB results showed that miR-9agomir significantly decreased the phosphorylated p65(p-p65)/p65,phosphorylated IKKβ(p-IKKβ)/IKKβ ratio associated with NF-κB signalling pathway in lung tissues,while miR-9 antagomir increased the p-p65/p65,p-IKKβ/ IKKβ ratio.(6)Effect of miR-9 on RUNX1 expression: qPCR,WB results showed that miR-9agomir significantly reduced the m RNAs and protein levels of RUNX1 in lung tissue,but miR-9 antagomir exerted the exact opposite effect.3.Mechanistic studies on the effects of miR-9 on alveolar procoagulation and fibrinolysis inhibition(1)With the extension of LPS treatment time,the expression level of miR-9 gradually decreased and reached the lowest at 24 h,and then gradually rebounded,while the expression of m RNA and protein level of RUNX1 increased and reached the maximum at 24 h,and then gradually down-regulated.(2)Effect of miR-9 on TF and PAI-1: Overexpression of miR-9(LV-miR-9)significantly inhibited the TF,PAI-1 m RNAs and protein levels in LPS-induced RLE-6TN cells,while low expression of miR-9(LV-miR-9-inhibitor)promoted the levels of the above indices.(3)Regulation of NF-κB signalling pathway by miR-9: LV-miR-9 significantly reduced the expression levels of p-p65/p65,p-IKKβ/IKKβ in LPS-induced RLE-6TN cells,while LV-miR-9-inhibitor exerted opposite effects on the expression of the above molecular protein ratios.(4)Targeted regulation of RUNX1 by miR-9: LV-miR-9 significantly reduced the m RNAs and protein levels of RUNX1 in LPS-induced RLE-6TN cells,but transfected with LV-miR-9-inhibitor,the m RNAs and protein levels of RUNX1 were significantly increased;furthermore,the double-win photoproteinase assay confirmed that RUNX1 was a miR-9 target gene.(5)Effect of RUNX1 on NF-κB signaling pathway: LV-RUNX1 significantly increased the expression of LPS-induced NF-κB signaling pathway-related proteins pp65/p65,p-IKKβ/ IKKβ in RLE-6TN cells,and CO-IP confirmed the interaction of IKKβ with RUNX1 protein.(6)miR-9 targets RUNX1 to regulate alveolar procoagulation and fibrinolysis inhibition: Rescue experiments showed that the expression of TF,PAI-1 and NF-κB signalling pathway were inhibited in RLE-6TN cells after transfection with LV-miR-9,while simultaneous transfection with LV-RUNX1 reversed the inhibition of the above indicators.Conclusions:1.miR-9 was significantly low expressed in the peripheral blood of ARDS patients,while RUNX1 m RNA was highly expressed,and the two were significantly negatively correlated in the peripheral blood of ARDS patients.2.miR-9 ameliorated LPS-induced lung injury in ARDS rats,reduced alveolar procoagulation and fibrinolysis inhibition in ARDS rats,and inhibited LPS-induced RUNX1 expression and NF-κB signaling pathway activation.3.miR-9 targets RUNX1 to regulate NF-κB signaling pathway to improve alveolar procoagulant and fibrinolytic inhibition in ARDS.