| Objective To study the molecular mechanism of ATGs through the miR-106a-related ceRNA network to regulate the expression of the negative regulatory factor SIRPα in TLRs,and then influence inflammatory effect on Mycobacterium tuberculosis in macrophages.Methods1.The expression of miR-106 a was detected by RT-PCR,SIRPα and inflammatory cytokines in inflammatory and autophagy models after treatment with THP-1 derived macrophages using LPS and rapamycin.2.Using H37 Ra infected THP-1 derived macrophages to detect the expression of miR-106 a at different infection time points and at different multiplicity of infection.3.MiR-106 a on TLRs negative regulatory factors SIRPα,autophagy-related gene ULK-1 and ATG16L1 were targeted by bioinformatics software analysis.And analysis of the sequence of its binding parts by miRbase,miRSVR and miRTarBase etc.To construct the plasmid of double luciferase reporter experiment,SIRPα 3’UTR,ATG16L1 3’UTR,and ULK-1 3’UTR and their mutants were cloned into pMIR-GLO plasmids,the targeted regulation of SIRPα,ATG16L1 and ULK-1 was verified by dual luciferase reporting system and Western blot,and the ATGs-3’UTR/miR-106a/SIRPα ceRNA network was demonstrated by dual luciferase experiments.4.A cell model of THP-1 derived macrophages infected with H37 Ra was constructed,and then the cells were transfected with miR-106 a mimics,miR-106 a inhibitor,miR-106 a mimics nc,and miR-106 a inhibitor nc respectively.SIRPαexpression detected by Western blot,immunofluorescence,and detection of theexpression of SIRPα and inflammatory cytokines by RT-PCR.Investigate the regulatory effect of miR-106 a on the inflammatory response process.5.ULK1 3’UTR and ATG16L1 3’UTR were cloned into pEGFP-C1 plasmid to construct pEGFP-ULK1 and pEGFP-ATG16L1 to achieve ATGs overexpression,synthesize ULK1 siRNA and ATG16L1 siRNA to achieve ATGs inhibition.They were transfected into a model that H37 Ra successfully infected THP-1 derived macrophages,the expression of SIRPα protein was detected by western blot and immunofluorescence,and the expression of SIRPα and inflammatory cytokines were detected by RT-PCR.To determine the regulation of autophagy-related gene ATGs for SIRPα,thus regulate the inflammatory response process.Results1.THP-1 derived macrophages were treated with LPS and rapamycin.RT-PCR results showed that the expression of miR-106 a in LPS group,miR-106 a inhibitor and LPS co-transfection group was significantly up-regulated compared with the control groups.miR-106 a expression was down-regulated in rapamycin group,miR-106 a mimics and rapamycin co-transfection group compared with control groups.The results of RT-PCR and Western blot showed that the expression of SIRPα groups was opposite to that of miR-106 a.And the results of RT-PCR showed that the expression trend of each subgroup of inflammatory cytokines was consistent with that of miR-106 a.2.Under the condition of H37 Ra infection,the RT-PCR results showed that the expression of miR-106 a was up-regulated(P<0.05)at different infection time points and different multiplicity of infection,in which the MOI is ten was very up-regulated(P<0.001).3.The miR-106 a,SIRPα,ATG16L1 and ULK-1 genes obtained by bioinformatics analysis were verified by double luciferase reporting system experiment have a targeting relationship.Transfection miR-106 a mimics significantly inhibited the relative luciferase activity of SIRPα,ATG16L1 and ULK-1WT,and their activity decreased(P<0.05).It was confirmed by Western blot that miR-106 a mimics can significantly inhibit the expression of SIRPα,ATG16L1 and ULK-1proteins,while the expression of SIRPα,ATG16L1 and ULK-1 proteins in the miR-106 a inhibitor group was significantly increased.The results showed that miR-106 a could inhibit SIRPα,ATG16L1 and ULK-1 expression at the posttranscriptional level.And also demonstrated the ATGs/miR-106a/SIRPα ceRNA network by double luciferase experiments.4.To demonstrate the effect of miR-106 a on inflammatory response,firstly through Western blot experiments,the results showed that the expression of SIRPα in the transfection miR-106 a mimics group was significantly reduced.Secondly,the miR-106 a mimics group showed a weaker green fluorescence compared to the transmembrane protein SIRPα of the nc group.Then the expression of SIRPα was detected by RT-PCR,the expression of SIRPα in miR-106 a mimics group decreased significantly,while the results in the miR-106 a inhibitor group were the opposite.The results suggested that miR-106 a can promote the process of H37Ra-mediated cellular inflammatory response by targeting inhibition of SIRPα expression.5.To investigate the effect of ATGs on inflammatory response,Western blot and cellular immunofluorescence experiments confirmed that the expression of negative regulatory factor SIRPα of inflammatory response increased in all pEGFP-ULK1 and pEGFP-ATG16L1 groups,and decreased in ULK1 siRNA and ATG16L1 siRNA groups.Finally,RT-PCR also confirmed the effect of ATGs inhibition of inflammatory response.The expression of negative regulatory factor SIRPα of inflammatory response increased in the pEGFP-ULK1 group,while the decrease of inflammatory cytokine expression indicated.The results in ULK1 siRNA and ATG16L1 siRNA groups were opposite.ATGs can compete for miR-106 a via the ceRNA network to inhibit cellular inflammatory responses.Conclusion1.LPS and rapamycin can change the expression of miR-106 a,SIRPα and inflammatory cytokines in THP-1 derived macrophages.miR-106 a can promote inflammatory response and inhibit cell autophagy.2.H37 Ra infected of macrophages can cause changes in the expression of miR-106 a and make it down-regulated.3.miR-106 a can inhibit the expression of SIRPα,ULK-1 and ATG16L1 genes,and confirm the existence of ATGs/miR-106a/SIRPα ceRNA network.4.miR-106 a can regulate the inflammatory response of macrophages against Mycobacterium tuberculosis by targeting SIRPα.5.ULK1 and ATG16L1 can compete with miR-106 a through ceRNA network to regulate the inflammatory effect of macrophages against Mycobacterium tuberculosis,thus achieving the effect of inhibiting cellular inflammatory response. |
