Mechanisms Of Sirp Alpha 3'UTR Regulating Macrophage Autophagy Against Mycobacterium Tuberculosis Through CeRNA Network

Objective To study the regulatory effect of SIRP alpha on several important autophagy genes ATG16L1,ATG7 and ULK-1 through competitive binding of micro RNA network to micro RNA-106 a,and to study the mechanism of cell autophagy against Mycobacterium tuberculosis(H37Ra).Method 1.Quantitative RT-PCR was used to detect the expression of mir-106 a and SIRP alpha in H37 Ra infected at different multiple or different infection times.2.Targeted regulation of autophagic genes ATG16L1,ATG7,ULK-1 and SIRP alpha related to inflammation pathway was obtained by using high-throughput gene expression database and bioinformatics software analysis.By comparing and analyzing the binding sequence of micro RNA-106 with SIRP alpha-3'UTR and ULK-1-3'UTR,ATG16L1-3'UTR and ATG7-3'UTR,we found that micro RNA-106 has the same binding sequence as SIRP alpha-3'UTR and ULK-1-3'UTR.The same seed region sequence,the sequence is 5'-AGCACUUU-3'.Therefore,we speculate that SIRP alpha-3'UTR may competently bind to micro RNA-106 a to regulate autophagy by regulating autophagy-related proteins through ce RNA networks.3.To construct double luciferase reporter experimental plasmids and clone wild-type SIRP alpha 3'UTR,ATG16L1 3'UTR,ATG7 3'UTR and ULK-13'UTR and their mutants into p MIR-GLO plasmids respectively.The targeting regulation effects of micro RNA-106 on SIRP alpha,ATG16L1,ATG7 and ULK-1 were verified by using double luciferase reporter system and Western blot.The SIRP alpha-3'UTR/micro RNA-106 a/ATGs ce RNA network was demonstrated by double luciferase assay.4.To construct H37Ra-infected THP-1-derived macrophage model,micro RNA-106 a mimic,micro RNA-106 a inhibitor and micro RNA-106 a NC were transfected into THP-1 cells respectively.The distribution of autophagic bodies and the expression level of autophagic-related proteins were detected by confocal laser microscopy,transmission electron microscopy and Western blot,and the structural characteristics of autophagic bodies were observed by electron microscopy to study the autophagic process of micro RNA-106 a.Regulatory role.5.SIRP alpha 3'UTR was cloned into p EGFP-C1 plasmid to realize the overexpression of SIRP alpha 3'UTR.Four groups of plasmids were co-transfected into the model of H37 Ra infected THP-1-derived macrophages.They were p EGFP-C1+micro RNA-106 a mimic,p EGFP-SIRP alpha+micro RNA-106 a mimic,p EGFP-SIRP alpha+micro RNA-106 a mimic,p EGFP-SIRP alpha+micro RNA-106 a minc.The distribution of autophagosomes and the expression level of autophagy-related proteins were detected by electron microscopy to determine the regulatory role of SIRP alpha in the autophagy process.Result 1.In the case of H37 Ra infection,the expression of SIRP alpha gene was significantly decreased by q RT-PCR,but the expression of mi R-106 a was first down-regulated and then up-regulated(P < 0.05).The expression of ATGs was significantly up-regulated,and ULK-1 was up-regulated to a very large extent(P < 0.0001).2.The double luciferase reporting system was used to validate the targeting relationship between the genes of mir-106 A and SIRPA,ATG16L1,ATG7 and ULK-1 obtained by bioinformatics analysis.The relative luciferase activity of SIRP alpha,ATG16L1,ATG7 and ULK-1wt was significantly inhibited by the transfection of micro RNA-106 a mimic,and its activity decreased by about one fold(P < 0.05).Western blot confirmed that the expression of SIRP alpha,ATG16L1,ATG7 and ULK-1 was significantly inhibited by micro RNA-106 a mimic,while the expression of SIRP alpha,ATG16L1,ATG7 and ULK-1 was significantly increased in the inhibitor group.The results showed that micro RNA-106 a could inhibit the expression of SIRP alpha,ATG16L1,ATG7 and ULK-1 at post-transcriptional level.The SIRP alpha-3'UTR/micro RNA-106 a/ATGs ce RNA network was also demonstrated by double luciferase assay.3.In order to demonstrate the effect of micro RNA-106 a on autophagic flow,Western blot experiments showed that the expression of autophagy-related proteins ATG16L1,ATG7 and ULK-1 decreased,the expression of LC3-II decreased significantly,the ratio of LC3II/LC3 I decreased,and the number of autophagic bodies in cellular immunofluorescence also decreased in the micro RNA-106 a mimic group.Transmission electron microscopy also confirmed that autophagy was small by observing the structure of autophagic bodies.The number of body decreased.The results of the micro RNA-106 inhibitor group were just the opposite.The results showed that the expression of ATG16L1,ATG7 and ULK-1 could be inhibited by micro RNA-106 a in H37Ra-mediated autophagy.4.In order to study the effect of SIRP alpha on autophagic flow,the expression of autophagy-related proteins ATG16L1,ATG7 and ULK-1 in all p EGFP-SIRP alpha and p EGFP-C1 groups increased,and the expression of LC3-II increased.Compared with the mimic + p EGFP-C1 group and the mimic + p EGFP-SIRP alpha group,the decreased autophagy of micro RNA-106 a mic could be increased by overexpression of SIRP alpha.Cellular immunofluorescence and transmission electron microscopy confirmed the role of SIRP alpha in promoting autophagy,suggesting that SIRP alpha can compete for micro RNA-106 a through ce RNA network to promote cell autophagy.Conclusion: 1.H37 Ra infection of macrophages can induce changes in the expression of SIRP alpha,micro RNA-106 A and ATGs.2.The expression of ULK-1,ATG16L1,ATG7 and SIRP alpha genes was inhibited by micro RNA-106 a,and the existence of SIRP alpha/micro RNA-106 a/ATGs ce RNA network was confirmed.3.Mi-106 a can regulate macrophage autophagy against Mycobacterium tuberculosis by targeting ATGs.4.SIRP alpha can compete with micro RNA-106 a via ce RNA network to regulate macrophage autophagy against Mycobacterium tuberculosis,thus promoting cell autophagy.