Molecular Mechanism Of Gga-miR-142-3p Targeting TAB2 To Inhibit Mycoplasma Gallisepticum Infection

Mycoplasma gallisepticum(MG)is a highly infectious pathogen.MG mainly adhere to chicken tracheal mucosal,cause chronic respiratory disease(CRD)and the damage of respiratory tract tissue.MG infection cause inappetence,lower weight gain and lower hatchability of Chicken eggs.Therefore,MG infection has caused huge economic loss to the poultry industry.So far,we haven't found any effective preventive measure to combat the disease.Thus,further study is needed to understand its pathogenesis and find new strategy to minimize economic damage.Studies have shown that micro RNAs(miRNAs)are abnormally expressed in various diseases.miRNAs participate in the regulatory network in host defence by mediating their target genes.Thus,miRNAs have great significance in the pathogenic diseases.Based on the results of previous high-throughput sequencing in our laboratory,this study aims to explore the role of gga-miR-142-3p and its target gene TAB2 in the MG infected chickens and provide a scientific basis for better understanding the pathogenic mechanism of MG.The relative expression of gga-miR-142-3p in SPF chicken embryo lung tissues and DF-1 cells,which is infected or not,was determined by quantitative real-time PCR(RT-q PCR)at different infection stages.Target gene of gga-miR-142-3p was predicted by bioinformatics analysis and proved by the dual-luciferase report system.The relative expression of TAB2 was detected when gga-miR-142-3p was up-regulated or down-regulated to confirm that TAB2 is the target gene of gga-miR-142-3p.We further used ELISA to detect the expression of proinflammatory cytokine interleukin-1?(IL-1?),interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-?)in DF-1 cells when gga-miR-142-3p or TAB2 is over-expressed or inhibited.Meanwhile,CCK-8 and flow cytometry were used to detect cell proliferation,cycle,and apoptosis,when miR-142-3p was overexpressed or inhibited.Results are as follows:1.The relative expression of gga-miR-142-3p in MG-infected chicken embryo lungs was significantly down-regulated on the 6th and 9th day after infection(p<0.01),and significantly up-regulated on the 12 th day after infection(p<0.05).In MG-1 infected DF-1 cells,the relative expression of gga-miR-142-3p was significantly down-regulated in the first 11 h(p<0.001),and significantly up-regulated in 26 h upon MG infection(p<0.05).2.The target gene of gga-miR-142-3p was predicted by bioinformatics database Targetscan and miRDB.The sequence conservation of target gene among species was analyzed.Combined with DAVID analysis,TAB2 is initially selected as the target gene of gga-miR-142-3p.3.The dual-luciferase results showed that in the gga-miR-142-3p mimics and WILD TAB2 3'UTR-psi CHECK2 plasmid co-transfection groups,the luciferase activities were significantly reduced.Whie,the gga-miR-142-3p inhibitor groups got the opposite results.The rescue test further confirmed that gga-miR-142-3p functions by binding with TAB2 3 'UTR,thereby further confirming that TAB2 is the target gene of gga-miR-142-3p.4.overexpression of gga-miR-142-3p significantly down-regulated the expression of TAB2 m RNA and protein levels(p<0.01),while gga-miR-142-3p inhibition significantly increased the expression of TAB2 m RNA and protein levels(p <0.01),further confirming that gga-miR-142-3p can negatively regulate the expression of TAB2,and TAB2 is the target gene of gga-miR-142-3p.5.TAB2 was significantly up-regulated on the sixth day upon MG infection(p <0.01),while TAB2 was significantly down-regulated on the ninth and twelfth day in MG-infected chicken embryo lung tissues(p<0.01).The expression of TAB2 was significantly higher than that of the control group in the first 15 h(p<0.05),and the expression of TAB2 was significantly lower than that in the control group at 26 h in MG-infected DF-1 cells(p<0.05).The results showed that the expression of gga-miR-142-3p was negatively correlated with the expression of TAB2.6.The expression of pro-inflammatory cytokines in the gga-miR-142-3p overexpression or si-TAB2 groups was significantly down-regulated,while the gga-miR-142-3p inhibition or TAB2 over-expression groups got the opposite results.Co-transfection with gga-miR-142-3p mimics and over-TAB2 plasmid can counteract the down-regulation of pro-inflammatory cytokines induced by gga-miR-142-3p over-expression.These results indicate that gga-miR-142-3p can down-regulate the expression of pro-inflammatory cytokines induced by MG infection via targeting TAB2.7.The activation of key proteins in NF-?B and MAPK signaling pathways were significantly down-regulated in gga-miR-142-3p over-expression or TAB2 knockdown groups(p < 0.05).In combination with the dual-luciferase results,it can be determined that miR-142-3p can inhibit the activation of NF-?B and MAPK signaling pathway to reduce the production of pro-inflammatory cytokines by down-regulating TAB2 expression.8.MG infection significantly disturbed the mitosis in DF-1 cells,inhibited the cell proliferation and promoted cell apoptosis.Over-expression of gga-miR-142-3p remarkably promoted cell proliferation at 24 h,48 h and 72 h upon MG infection,increased the portion of cells remained in S and G2 phases,and inhibited the cell apoptosis.While,inhibition of gga-miR-142-3p inhibited cell proliferation,increased the portion of cells remained in G1 phases,promoted cell apoptosis.Our research shows that up-regulation of gga-miR-142-3p upon MG infection can reduce the production of pro-inflammatory cytokines by negatively regulating NF-?B and MAPK signaling pathways via targeting TAB2.gga-miR-142-3p can promote cell proliferation,cell cycle and inhibited cell apoptosis to defense against MG infection.