The Role And Mechanism Of MiRNA-130a-3p In Inflammatory And Fibrotic Phases Of Pulmonary Fibrosis In Mice

Objective: Pulmonary fibrosis(PF)is a complication of respiratory system that seriously harms human health and is the common outcome of chronic lung diseases caused by many reasons.With the aggravation of the disease,the respiratory function of the patient continues to deteriorate,and its morbidity and mortality increase.Although lung transplantation can improve the quality and prolong life in PF patients,its application is limited by the shortage of donated organs,rejection reactions,and infections.Bleomycin(BLM)induced PF animal models were most similar to human PF histopathological changes.After intrattracheal administration of BLM into animals,acute lung injury was first induced,characterized by apoptosis of alveolar epithelial cells,invasion of inflammatory cells and secretion of pro-inflammatory cytokines.This was followed by increasing markers of fibrosis,fibroblasts were activated,and the release of excess extracellular matrix,while cytokines amplified the inflammatory response and further triggered fibroblast proliferation and differentiation.We found abnormal expression of miR-130a-3p in PF patients through bioinformatics analysis.Meanwhile,our previous study found that miR-130a-3p targeted transforming growth factor β receptor(TGFBR)II affected the proliferation and differentiation of lung fibroblasts.At the same time,it could reduce the apoptosis and inflammatory reaction of alveolar epithelial cells.However,the mechanism of miR-130a-3p on the pathological development of PF is rarely studied.This study aims to explore the expression levels and potential therapeutic effects of miR-130a-3p at inflammatory and fibrotic phases of PF,and further study the mechanism of miR-130a-3p in the main effector cells of PF.Single-cell transcriptome sequencing technology was used to analyze the data of BLM-induced PF total lung single cells to identify the main effector cells,and the main effector cells of PF were identified to explore the mechanism of m RNA-130a-3p in PF.Methods:The effects of miR-130a-3p on BLM-induced PF in mice at inflammatory and fibrotic phases:1.BLM-induced PF model,q RT-PCR and Western Blot were used to verify the expression of genes related to inflammation and fibrosis to clarify the inflammatory and fibrotic phases of PF.2.Bioinformatics analysis of miRNAs expression in PF.3.The expression levels of miR-130a-3p at inflammatory and fibrotic phases of PF were detected.4.Establishment of PF models: PF models in inflammatory and fibrotic phases were established,and miR-130a-3p agomiR was injected into tail vein of mice.The changes of lung morphology,body weight,lung wet-dry weight ratio,and lung index were detected.HE and Masson staining were used to detect the pathological changes.5.The expression of miR-130a-3p in the lung tissues of mice: q RT-PCR and in situ hybridization were used to study the expression of miR-130a-3p in the lung.6.Effects of miR-130a-3p on inflammatory phase of PF: Cytokine secretion level and exudation of inflammatory cells in BALF were detected by ELISA and Wright-Giemsa staining.Western Blot and q RT-PCR were used to detect the changes of NF-κB signaling pathway related proteins and m RNA.7.Effects of miR-130a-3p on fibrotic phase of PF: HYP content in lung tissue was measured by alkaline hydrolysation.Western Blot and q RT-PCR were used to detect the changes of TGF-β/Smad signaling pathway related proteins and m RNA.The expression of α-SMA positive cells on the surface of myofibroblasts was verified by immunofluorescence staining.The sc RNA-seq analysis of BLM-induced mouse PF model:1.The whole lung single-cell transcriptome data of BLM-induced PF at inflammatory and fibrotic phases were analyzed to identify the main effector cell types.2.Differential gene expression profiles were established and functional enrichment analysis was performed to explore the pathogenesis of PF.In vitro verification of the effects of miR-130a-3p on inflammation and fibrosis:1.Western Blot and q RT-PCR assay were used to detect the activation of NF-κB signaling pathway and the expression of miR-130a-3p in MH-S cells treated with LPS and inhibitors at different concentrations/time.2.Western Blot assay was performed to determine the effects of miR-130a-3p mimics and inhibitors on inflammatory responses.3.After MRC-5 cells were treated with different concentrations/time of TGF-β1 and inhibitors,the expressions of miR-130a-3p and fibrosis markers were detected by Western Blot and q RT-PCR assay.4.The effects miR-130a-3p mimics and inhibitor on proliferation and differentiation of MRC-5 were determined by CCK-8,Western Blot and immunofluorescence assay.5.miR-130a-3p directly targeting TGF-βRII was detected by dual luciferase reporter gene.6.TGF-βRII in MRC-5 was specifically knocked down by small interfering RNA,and the effects of TGF-βRII on fibrosis markers were determined by Western Blot.Results:The effects of miR-130a-3p on BLM-induced PF in different periods:1.On day 7 after BLM model establishment,inflammatory markers TNF-α,IL-1β and IL-6 were highly secreted,P65/p-IκB were activated,and IκB expression was inhibited.However,the expression of fibrosis markers TGF-β1,α-SMA,FN and TGF-βRⅡincreased gradually.The expression of epithelial marker E-cadherin was decreased.2.Bioinformatics analysis showed that miR-130a-3p was differentially expressed in PF patients.3.In mouse PF model,miR-130a-3p expression increased on day 7 and decreased on day28.4.Establishment of miR-130a-3p treated mouse PF model: In the inflammatory phase,the body weight of the mice decreased significantly.In the fibrotic phase,the body weight decreased rapidly.However,after the administration of miR-130a-3p,the degree of weight loss was reduced.The lung tissues were pink,shiny,uniform in color and sharp in edge,without blood stasis,pechymosis,discoloration and swelling.Lung tissue was swollen and hyperemia in inflammatory phase.In the fibrotic phase,both lungs were swollen and enlarged.Deep red hemorrhagic spots were diffused in both lungs,and obvious blood stasis and edema appeared on the lung surface.In the miR-130a-3p treatment group,the volume was slightly enlarged and the gloss was common,with occasional scattered hemorrhagic spots,but the degree of blood stasis and edema was reduced,the texture was slightly rough,and the lung edge was sharp.Lung wet-dry weight ratio,lung index and total lung water content increased in the BLM group during inflammatory and fibrotic phase,while lung edema was significantly improved after treatment with miR-130a-3p,especially during inflammatory phase.Compared with the BLM group,the alveolitis score and fibrosis score in the lung tissue pathological sections of mice treated with miR-130a-3p were lower.5.Expression of miR-130a-3p in lung tissues of mice: The expression of miR-130a-3p in the BLM group was increased in the inflammatory phase and decreased in the fibrotic phase.miR-130a-3p in lung tissues was significantly expressed after the injection,which illustrated that intervention was successful.6.Effects of miR-130a-3p on inflammatory phase of PF mice: The secretion levels of cytokines TNF-α,IL-1β and IL-6,the total number of cells,the number and percentage of neutrophils and lymphocytes in BALF of BLM group increased,while the secretion of cytokines and the number of cells in the BLM + miR-130a-3p group decreased.The protein or m RNA expressions of IL-6/1β,P65,p-IκB,TNF-α,CD14 and CD68 in BLM group were up-regulated,while the expressions of IκB and CD19 were down-regulated compared to the Control.The above indicators were reversed after miR-130a-3p treatment.7.Effects of miR-130a-3p on fibrotic phase of PF mice: The TGF-β1 level and HYP content of BLM group increased,whereas decreased after miR-130a-3p treatment.The protein or m RNA expressions of α-SMA,FN,Col Ⅰ/Ⅲ,TGF-β1,TGF-βRⅠ/Ⅱ,Smad4 and the phosphorylation level of Smad2/3 protein in BLM group were significantly increased.However,the expression was decreased after miR-130a-3p treatment.The expression of E-cadherin was contrary to the above.8.Immunofluorescence staining results showed that BLM increased α-SMA positive cells in lung tissues at inflammatory and fibrotic phases,and decreased α-SMA positive cells after treatment with miR-130a-3p.The sc RNA-seq analysis and validation of BLM-induced mouse PF model:1.We annotated the cells into 13 types of cells,and found that the number of macrophages and fibroblasts increased in both inflammatory and fibrotic phases,while the number of alveolar epithelial cells decreased.2.We successfully established the differential gene expression of macrophages,fibroblasts and epithelial cells,among which different genes were enriched in immune cell activation,response to injury and external stimuli,and regulation of TNF-α production.Whereas those in fibroblasts were concentrated in the response to injury,the formation of extracellular matrix and structure,and the biosynthesis of ribosomes.The effects of miR-130a-3p on inflammation and fibrosis in vitro:1.With the increase of LPS treatment concentration and time,miR-130a-3p,P65,p-IκB and TNF-α increased,and the expression of IκB decreased.10 ng/m L LPS was administrated for 12 h as the follow-up treatment conditions.2.NF-κB inhibitor blocked the effect of LPS on miR-130a-3p expression.3.After transfection with miR-130a-3p mimics,the expression of TNF-α protein was inhibited.4.With the increase of TGF-β1 concentration and treatment time,the expression level of miR-130a-3p decreased,α-SMA,FN,and Col Ⅰ/Ⅲ increased.10 ng/m L TGF-β1 was administrated for 24 h as the follow-up treatment conditions.5.Inhibitors of TGF-βRI/II and Smad3 blocked the inhibitory effect of TGF-β1 on miR-130a-3p.These results indicated that TGF-β1 reduced the expression level of miR-130a-3p by acting on TGF-βRI/II/Smad3 pathway.6.After miR-130a-3p overexpression,the proliferation and differentiation ability of MRC-5 cells were inhibited,and the protein levels of α-SMA,FN and TGF-β/Smad pathway were decreased.Similarly,after TGF-βRII knockdown,the expression of the above indicators was also inhibited.7.miR-130a-3p could directly target TGF-βRII,thereby inhibiting its expression.Conclusion: In BLM-induced PF model,macrophages were activated,and the expression of NF-κB pathway genes and miR-130a-3p was up-regulated in inflammatory phase.Fibroblast proliferation,EMT,and fibrosis related genes were up-regulated,and miR-130a-3p expression was down-regulated in fibrotic phase.High expression of miR-130a-3p in vivo could effectively alleviate BLM-induced lung injury in inflammatory and fibrotic phases.miR-130a-3p could alleviate inflammatory response by inhibiting TNF-αto affect NF-κB pathway,while targeting TGF-βRII and interfering with TGF-β/Smad signaling pathway to affect TGF-β1-induced phenotypic transformation of lung fibroblasts.This provides an experimental basis for miR-130a-3p in the future targeted therapy of PF.