| Background: Paraquat(PQ)poisoning is common in clinical diagnosis and treatment of emergency.After acute PQ poisoning,it can be distributed in various organs,but the irreversible and extensive pulmonary fibrosis is the fatality cause in PQ patients.Paraquat(PQ)mainly causes irreversible damage to the body through oxidative stress,inflammatory response,hypoxia,DNA and mitochondrial damage leading to apoptosis,affecting cell signal transduction(Notch1pathway,Wnt-signaling pathway),cytokines(TGF-A,IL-6,TGF-P,IL-33,etc.)and other pathways.Studies have been found that EMT is involved in the PQ-induced pulmonary interstitial fibrosis.At present,the treatment for pulmonary interstitial fibrosis caused by PQ poisoning is blank.Therefore,it is of great important to find advanced therapeutic targets.Recent studies have found that microRNA(miRNA)can partially complement and bind with target genes(circ RNA,pseudogene,lnc RNA,m RNA,etc.)through microRNA response elements(MREs)to inhibit the expression of target genes,then regulates the process of cell via a variety of signaling pathways and participates in the occurrence of apoptosis,inflammation and EMT.Many studies have proved that numerous miRNAs are participated in the occurrence and development of pulmonary fibrosis,such as Let-7d,miR-29,miR-21,miR-34 a,etc.Regulator of G-protein signalling2(RGS2)is an inhibitor of G-protein coupled protein,involved in the regulation of blood pressure and nerve function,as well as the hypersecretion of mucin induced by acid in airway epithelial cells.Pirfenidone(PFD)is a molecular drug with anti-fibrosis and anti-inflammatory activity,which can relieve pulmonary interstitial fibrosis in patients with IPF.Some studies have proved that up-regulated RGS2 was one of the important mechanisms of PFD in the treatment of pulmonary interstitial fibrosis.The high-throughput results of our group showed that the newly constructed miR-9-8974-5p and RGS2 constituted a regulatory network.However,whether this predicted network is involved in pulmonary EMT caused by PQ poisoning has not been reported so far.Whether miR-9-8974-5p could participate in pulmonary EMT induced by paraquat via regulating RGS2.Whether pirfenidone has a potential therapeutic effect on pulmonary EMT induced by paraquat or whether the therapeutic effect is regulated by mir-9-8974-5p/RGS2 pathway.Objective: To find the potential regulatory genes in pulmonary EMT by PQ-induced and provide potential therapeutic targets.To study the feasibility and possible mechanism of PFD in the treatment of pulmonary interstitial fibrosis caused by paraquat poisoning.Methods:1.C57/BL6 mice were used to constructed the animal model of PQ-induced EMT.The pulmonary fibrosis was observed at histopathological level in mice by HE staining and Masson staining.The relative expression of E-cadherin and α-SMA was detected to verify the occurrence of EMT in the model mice via Western-blot and qRT-PCR.2.Transcriptome high-throughput sequencing was used to analyze the changes in m RNA and non-coding RNA(miRNA,circ RNA and lnc RNA)transcription levels in lung tissues with PQ-induced EMT,and correlation analysis was conducted by bioinformatics methods.3.The suitable exposure concentration of PQ poisoning was studied by CCK8 experiment,and the cell model of PQ poisoning was constructed.The relative expression of the E-cadherin、α-SMA and Vimentin was detected via qRT-PCR and Western-blot,the ability of cell migration was detected via Transwell experiment,to verify that the PQ virus causes EMT.4.In the animal and cell model,WB and qRT-PCR were used to detect the change of RGS2 at the protein and gene level.qRT-PCR was used to verify the expression changes of miR-9-8974-5p at the RNA level in animal and cell models.5.After transfection with mimic or inhibitor miR-9-8974-5p,the expression of miR-9-8974-5p was up-regulated or down-regulated.qRT-PCR and Western-blot were used to test the effects of up-regulation or down-regulation of miR-9-8974-5p on the expression of E-cadherin,α-SMA,Vimentin and RGS2.The regulation of miR-9-8974-5p on RGS2 expression after PQ induced was further verified.6.To detect whether RGS2 was the target of miR-9-8974-5p by using Luciferase reporter assay in the cell model.The p SI-Check2-RGS2-WT and p SI-Check2-RGS2-MUT were constructed in cell and co-transfected miR-9-8974-5p with the mimic or mimic NC.The relative activity of luciferase in each group was compared.7.Pirfenidone was added into the MLE-12 cell and animal model of EMT induced by PQ poisoning.The expression of E-cadherin,α-SMA and RGS2 were verified via qRT-PCR and Western-blot,respectively.Transwell experiment was used to test the migration ability of cells after PFD treatment.8.The expression of miR-9-8974-5p was contrived up or down-regulated after transfection with miR-9-8974-5p mimic or inhibitor in cell model.The relative expressions of E-cadherin,α-SMA and RGS2 in MLE-12 cells were measured by qRT-PCR and Western-blot at gene level and protein level after 0.05mg/ml PFD treatment,while researched the effect of the expression of the PFD to alleviate the function of the EMT.Results:1.HE stain of lung tissues of mice exposed to PQ(30mg/kg)showed that lung tissue structure was obviously destroyed,alveoli ruptured,and alveolar septa incrassated;The Masson stain showed that a large amount of collagen deposition appeared in lung tissues which was induced by PQ.Pathological results showed that pulmonary fibrosis occurred in mice after PQ exposing.Western-blot results showed that E-cadherin expression in PQ group was down-regulated(P<0.05),while α-SMA expression was up-regulated(P<0.05),indicating that EMT occurred in tissues;The results of qRT-PCR were consistent with those results of Western-blot(P<0.05).2.The result of high-throughput sequencing: After PQ induced,We found only three miRNAs interacting with circ RNAs changes.Thereafter,we found that 337 circ RNAs were correlated with m RNA.We analysed the relationship between 1070 pairs of miRNA and lnc RNA and found that 159 pairs were negatively correlated.We analysed the relationship between 2150 pairs of miRNA and m RNA in the PQ-induced lung injury group and found that there were 265 pairs of miRNA-m RNA negatively correlated.3.48.2% of MLE-12 cells survived at the concentration of 200μmol/L in PQ-induced group through CCK8,and qRT-PCR results showed that E-cadherin expression in PQ-induced group were down-regulated(P<0.05),while Vimentin and α-SMA was up-regulated(P<0.05);The results of Western-blot were the same as those of qRT-PCR(P<0.05);The ability of migration was enhanced in PQ-induced cells via Transwell(P<0.05).4.In the animal and cell model,the expression of RGS2 was significantly down-regulated(P<0.05),while the expression of miR-9-8974-5p was significantly up-regulated via qRT-PCR(P<0.05).5.In cell model,the expression of miR-9-8974-5p,α-SMA and Vimentin(P<0.05)were significantly increased with mimic,and the expression of RGS2 and E-cadherin was decreased(P<0.05).While the inhibitor could down-regulate the expression of miR-9-8974-5p,α-SMA and Vimentin(P< 0.05),the expression of RGS2 and E-cadherin were up-regulated(P< 0.05).6.In the cell model,the Luciferase activity of p SI-Check2‐RGS2‐WT+ mimic was significantly decreased than its NC group(P<0.05).And the luciferase activity of p SI-Check2‐RGS2‐MUT+mimic had no difference with its NC group(P>0.05).The miR-9-8974-5p can negatively regulate RGS2.7.In animal model and cell model,the intervention of PFD group was found that the expression of E-cadherin and RGS2 were up-regulated(P<0.05),while miR-9-8974-5p and α-SMA were down-regulated(P<0.05);Transwell experiment showed that the migration ability was reduced in PFD group(P<0.05).8.In the cell model with PFD therapy,the expression of miR-9-8974-5p and α-SMA was up-regulated by the addition of mimic miR-9-8974-5p(P<0.05),and the expression of RGS2 and E-cadherin was decreased(P<0.05),while the expression of miR-9-8974-5p and α-SMA was down-regulated by the addition of inhibitor miR-9-8974-5p(P<0.05),and the expression of RGS2 and E-cadherin were up-regulated simultaneously(P<0.05);It was proved that miR-9-8974-5p has a regulatory effect on PFD in the treatment of PQ-induced EMT.Conclusion: miR-9-8974-5p can mediate the pulmonary EMT PQ-induced by inhibiting RGS2.PFD has a potential therapeutic effect on PQ-induced pulmonary EMT through the regulation of miR-9-8974-5p /RGS2. |
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