Researches On The Regulation Of TLR4/MyD88 Signaling Pathway By MiR-29a

Objective: Mycobacterium tuberculosis(MTB)is a pathogen that causes tuberculosis(TB),which is mainly parasitic in macrophages after infection.Macrophages are members of the innate immune system.The pattern recognition receptors(PRRs)are insist on the surface of the cell membrane and on the Intracellular endometrial membrane,which can identify pathogens.After the pathogens recognized by the PRR,the signaling pathway of immune response will be activated through the cascading of the proteins,which can produce cytokines.Then the adaptive immune response will be activate,which make body forming the complete immune response network to eliminate pathogens effectively.This experiment is revealing whether the Toll-like receptor 4/myeloid differentiation factor 88(TLR4/MyD88)signaling pathway was regulated by microRNA-29a(miR-29a)after Bacillus Calmette-Guerin Vaccine(BCG)be infected with RAW264.7 cells.It can provide an experimental basis for the pathogenesis of MTB and the discovery of drug targets.Methods: Six experimental groups were set up in this experiment,including blank control group,miR-29 a inhibitor vector transfection group,negative control transfection group,BCG infection group,miR-29 a inhibitor vector transfection + BCG infection group,and negative control + BCG-infected group.We detected the expression of mRNA by real-time PCR in TLR4/MyD88 signaling pathway,including of TLR4,MyD88,TRAF6,MyD88,AP-1,interleukin-6(IL-6),interleukin-12(IL-12),and Tumor Necrosis Factor-α(TNF-α);and the expression of total protein and phosphorylated protein of Mitogen-activated Protein Kinase Kinase 4(MAP2K4)and Transcription Activitor-1(AP-1)by Western blot in TLR4/MyD88 signaling pathway,;and the expression levels of the cytokines IL-6,IL-12 and TNF-α were measured by Enzyme-linked immunosorbent assay(ELISA).Results: The expression levels of miR-29 a and the elated genes(TLR4,MyD88,TRAF6,MAP2K4,AP-1,IL-6,IL-12 and TNF-α)in TLR4/MyD88 signaling pathway were both up-regulated after BCG infected in macrophages in 24 hours.With the macrophages infected by BCG after transfected by miR-29 a inhibitory vector,we find out that the expression levels were positively correlated between the mRNA of TLR4,MyD88,TRAF6,MAP2K4,AP-1,IL-6,IL-12 and miR-29 a,but the expression levels were negatively correlated between the mRNA of TNF-α and miR-29 a by real-time PCR.It was not correlated between the expression levels of MAP2K4,AP-1 and miR-29 a by Western blot.And the result of detected by ELISA were also not correlated between the expression levels of cytokines IL-6,IL-12,TNF-α and miR-29 a.Conclusion: The expression level of miR-29 a and the mRNA of genes in TLR4/MyD88 signaling pathway was related.But miR-29 a and the protein level has no correlation.