ITGA11 Functions As CeRNA To Regulate MMP2 In Paraquat Induced Epithelial-mesenchymal Transition

Background: Paraquat(PQ)is one of the most common poisons in clinical practice.In clinical emergency cases,the fatality rate caused by accidental or intentional ingestion of PQ is extremely high.PQ intoxication in any exposure pathway is mainly concentrated in the lung.The irreversible and extensive pulmonary fibrosis is the leading cause of death.Epithelial-mesenchymal transition(EMT)is involved in pulmonary interstitial fibrosis induced by PQ poisoning.During this process,the expression of epithelial markers such as epithelia cadherin(E-cadherin)are downregulated,while the expression of mesenchymal markers such as ?-smooth muscle actin(?-SMA)and Vimentin are increased,cells connections are weakened or disappeared,extracelluar matrix(ECM)is accumulated and the abilities to migrate and invade are acquired.At present,there is no effective treatment for pulmonary interstitial fibrosis caused by PQ poisoning,so it is of great significance to find new therapeutic targets.In recent years,more and more attention has been paid to the functions of nonprotein-coding RNA(nc RNA)in cellular pathophysiological regulation.Non-coding RNA can be divided into housekeeping RNA and regulatory RNA,and regulatory noncoding RNA includes mi RNA,Lnc RNA,si RNA,pi RNA and so on.Non-coding RNA can regulate cellular processes through a variety of signaling pathways,many of which are involved in inflammation,EMT,etc.The competing endogenous RNA(ce RNA)network refers to that some RNAs with the same mi RNA response element(MRE)can competitively bind to the same mi RNA locus to reduce the inhibiton of mi RNA on other target,thus influencing biological behavior.Studies have proven that MMP2,a member of the Matrix metalloproteinase(MMP)family,is involved in abnormal tissue remodeling during PQ induced pulmonary fibrosis.The Integrin alpha 11(ITGA11)may be involved in the process of pulmonary fibrosis and the mi R-17-5p is decreased in idiopathic pulmonary fibrosis(IPF)lung tissues and fibroblasts.The ENCORI website has predicted that ITGA11 could competitively combine mi R-17-5p as an ce RNA of MMP2.However,whether this predicted ce RNA network is involved in pulmonary EMT caused by PQ poisoning has not been reported so far.Objective: To reveal the potential ce RNA regulatory network in pulmonary EMT induced by PQ poisoning and provide potential therapeutic targets for EMT induced by PQ poisoning.Methods: 1.We used C57/BL6 mice to constructed in vivo model of PQ induced EMT by intraperitoneally injecting with 30mg/kg PQ.HE staining and Masson staining were used to observe whether the mice developed pulmonary fibrosis at the histopathological level.q RT-PCR was used to check the relative expressions of E-cadherin and ?-SMA to verify whether mice underwent EMT in this model.2.q RT-PCR was used to verify the relative expressions of ITGA11 and MMP2 in the lung tissues after PQ exposure at the m RNA level.3.Explored the appropriate dose of chronic PQ poisoning in A549 cells and constructed the cell model of pulmonary EMT induced by PQ poisoning.q RTPCR was used to verify the relative expressions of E-cadherin,?-SMA and MMP2 in the experimental group at the m RNA level,and western-blot was used to verify the relative expressions of E-cadherin,?-SMA and MMP2 in the experimental group at the protein level.The changes of cell migration ability in PQ group was verified by wound healing assay and transwell assay.Immunofluorescence was used to further detect the changes of E-cadherin and ?-SMA expression in the cell model.4.The expression changes of ITGA11 and mi R-17-5p in the cell model were verified by q RTPCR from the gene level.5.Used specific si RNA to down-regulate the expression of ITGA11,q RT-PCR was used to detect of effect of si RNA sequence and to detect the expression of MMP2 after down-regulation of ITGA11,western-blot was used to explore the influence of down-regulated ITGA11 on MMP2 and E-cadherin after PQ exposure.The effect of down-regulated ITGA11 on MMP2 expression after PQ exposure was further verified by immunofluorescence.6.The expression of mi R-17-5p was artificially up-regulated or down-regulated by mimic or inhibitor of mi R-17-5p to test the effect of mi R-17-5p on ITGA11 and MMP2.q RT-PCR was used to verify the effect at the gene level.Immunofluorescence was used for further verification after PQ exposure.7.Luciferase reporter assay was used to detect whether ITGA11 was the target of mi R-17-5p.We constructed the wide type(WT)or mutated(MUT)p SICheck2 dual?luciferase reporter vector and co-transfected with the mimic or mimic NC of mi R-17-5p.Then compared the relative luciferase activities between different groups.Results: 1.The HE staining results revealed that the PQ treated group showed obvious destruction of pulmonary structure,alveolar ruptured and fused and thickened interalveolar septum.The Masson staining results showed significant increase in deposition of collagen fibres in PQ group than normal control group.q RT-PCR results showed that the expression of E-cadherin was down-regulated(P<0.05)and expression of ?-SMA was up-regulated in mice model of PQ poisoning(P<0.05).2.q RT-PCR results showed that the expression of ITGA11 and MMP2 was up-regulated in mice model of PQ poisoning(P<0.05).3.The survival rate of A549 cells was still better when the PQ concentration reached 60?mol/L.q RT-PCR results showed that the expression of Ecadherin was down-regulated(P<0.05)and expression of ?-SMA and MMP2 was upregulated(P<0.05)in PQ treated cells.Western-blot results showed that the expression of E-cadherin was down-regulated(P<0.05)and expression of ?-SMA and MMP2 was up-regulated(P<0.05)in PQ treated cells.The immunofluorescence results were consistent with western blot results.The wound healing assay and transwell assay showed that the migration ability of PQ treated cells was enhanced(P<0.05).4.The expression of ITGA11 was up-regulated(P<0.05)and the expression of mi R-17-5p was down-regulated(P<0.05)in PQ treated cells.5.q RT-PCR results showed that the si RNA sequence of ITGA11 could down-regulate the expression of ITGA11(P<0.05)and MMP2 was down-regulated after the down-regulation of ITGA11(P<0.05).The western-blot results showed that the expression of MMP2 was decreased and the expression of E-cadherin was increased in PQ treated group after the down-regulation of ITGA11(P<0.05).Immunofluorescence results showed that the expression of MMP2 was also down-regulated after ITGA11 down-regulated by si RNA in both nonPQ treated and PQ treated group,and the fluorescence intensity of ITGA11 and MMP2 was increased after PQ exposure.6.q RT-PCR results showed that the mimic could significantly increase the expression of mi R-17-5p(P<0.05)and the expression of ITGA11 and MMP2 was down-regulated simultaneously with the up-regulation of mi R-17-5p by transfection of its mimic(P<0.05),while the inhibitor could decrease the expression of mi R-17-5p(P<0.05)and expression of ITGA11 and MMP2 was up-regulated simultaneously with the down-regulation of mi R-17-5p by transfection of its inhibitor(P<0.05).The immunofluorescence results in both non-PQ treated group and PQ group were consistent with q RT-PCR results and the fluorescence intensity of ITGA11 and MMP2 was increased after PQ exposure.7.The luciferase activity of p SICheck2?ITGA11?WT+mimic was lower than that of p SI-Check2?ITGA11?WT+mimic NC(P<0.05).And the luciferase activity of p SI-Check2?ITGA11?MUT+mimic had no difference with that of p SI-Check2?ITGA11?MUT+mimic NC(P>0.05).In other word,the ITGA11 was the target of mi R-17-5p.Conclusion: ITGA11 functions as ce RNA to regulate MMP2 in PQ induced pulmonary EMT.