A Study About The Regulatory Of Effects Of Mmu-miR-25-3p/DUSP10 Signaling Pathway On Autophagy And Intracellular Clearance Of Bcg In Bcg-infected Macrophages

Objective The key differential exosome miRNA that regulate autophagy in macrophages infected with BCG were screened by bioinformatics analysis,and its regulatory effects on autophagy and intracellular clearance of BCG were verified by in vitro cell experiments.Methods In this study,Mycobacterium tuberculosis bacillus Calmette-Guerin(BCG)was used to infect macrophage Raw264.7 to establish an in vitro cell experimental model,and then the exosomes in the culture supernatant of the infected and uninfected groups were separated and collected by ultracentrifugation.Afterwards,transmission electron microscopy used to observe the morphological structure of exosomes,nanoparticle tracking analysis used to detect particle size of exosome,and the surface marker proteins CD9 and CD81 were detected by Western blotting.The exosomal RNA found in the previous high-throughput sequencing was randomly sampled by RT-q PCR technology.Through enrichment analysis and Hub gene analysis,key differentially expressed miRNA and its target genes were selected.The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the Target Scan database,and this was further verified by dual luciferase reporter gene assay.The optimal observation time point of autophagy was determined by detecting the expression level changes of mmu-miR-25-3p,DUSP10 and LC3-II at 0,1,4,8,12,24,48 and 72 h after infection with BCG.After designing and synthesizing mmu-miR-25-3p mimics,mmu-miR-25-3p inhibitor,si-DUSP10,miR-NC and si-NC,they were transfected into macrophage Raw264.7 in groups,and PD98059(ERK Inhibitor)group was observed together.After that,the expression levels of DUSP10 and p-ERK1/2 were detected at the optimal observation time point,and the expression levels of LC3-II,beclin1,Atg5 and Atg7 were detected.The autophagy flux of macrophage Raw264.7in each group was observed by confocal laser microscopy,and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy.Finally,the intracellular BCG load of macrophage Raw264.7 was evaluated by colony forming unit assay(CFU).Results The exosomes extracted in this study meet the various standards for exosome characterization,and the expression trend of 6 exosomal miRNAs randomly sampled is consistent with the high-throughput sequencing results of exosomal miRNAs in previous studies.Enrichment analysis and Hub gene analysis indicated that mmu-miR-25-3p is a key differentially expressed miRNA for autophagy regulation in Raw264.7 macrophages infected with BCG,and its target gene may be DUSP10.The search of the Target Scan database and the verification of the dual luciferase reporter gene assay results showed that DUSP10 is the target gene of mmu-miR-25-3p.By observing the expression changes of mmu-miR-25-3p and DUSP10 over time after BCG infected macrophage Raw264.7,it was determined that 8 h after BCG infection was the best observation time point for observing autophagy.Compared with the control group,transfection of mmu-miR-25-3p mimics and si DUSP10 could significantly down-regulate the expression levels of DUSP10 m RNA and protein(p<0.05),and up-regulate the expression levels of p-ERK1/2,LC3-II,beclin1,Atg5 and Atg7(p<0.05).The expression distribution of DUSP10 was observed by immunoelectron microscopy,which further confirmed the above results.The observation of autophagy flox and transmission electron microscopy further proved that transfection of mmu-miR-25-3p mimics and si-DUSP10 can enhance the autophagy level of macrophage Raw264.7 infected with BCG.Compared with the control group,the count of CFU in macrophage Raw264.7 transfected with mmu-miR-25-3p mimics and si-DUSP10 was significantly reduced(p<0.05).Conclusion mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10,and enhances the autophagy of macrophage Raw264.7 infected with BCG,thereby promoting the clearance of intracellular BCG and reducing the intracellular BCG load.mmu-miR-25-3p is a new potential target for anti-tuberculosis immunomodulatory therapy.