MiR-509-5p Regulates Anti-infection For Mycoplasma Pneumonia In Sheep Through Targeting NF-?B Pathway

Objective:In order to study the molecular mechanism of anti-mycoplasma pneumonia in sheep,we screened the differentially expressed microRNAs(miRNAs)and genes related to Manna-binding lectin(MBL)anti-mycoplasma pneumonia in sheep by bioinformatics methods,determined the biological functions of target genes,and verified them at the cellular level,in order to explore deeply that their regulatory mechanism in Mycoplasma pneumonia in sheep,so as to enrich the anti-mycoplasma pneumonia in sheep,the molecular mechanism of inflammation lays the foundation.Methods:(1)miRNA may play a key role in MBL anti-mycoplasma pneumonia in sheep.Bioinformatics methods were used to analyze the Small RNA-seq database and RNA-seq database established in the previous study of this group to screen differentially expressed miRNAs and genes.Software such as TargetScan predicts target genes that differentially express miRNAs.(2)The wild-type and mutant reporter genes containing the target gene TRAF6 3' UTR region were constructed,and the target regulation of miR-509-5p on the target gene TRAF6 was verified by the Dual-Luciferase? Reporter Assay System.(3)Respiratory mucosa epithelium is a key host of Mycoplasma pneumoniae.By primary culture of sheep respiratory mucosal epithelial cells,miR-509-5p mimics,miR-509-5p mimicsNC,miR-509-5p inhibitor and miR-509-5p Inhibitor NC was transfected into the cells,and qRT-PCR was used to analyze the expression of miR-509-5p in the expression of target gene TRAF6 and NF-?B signaling pathway.(4)Transfected the miR-509-5p mimics and its negative control of susceptible sheep respiratory mucosal epithelial cells,and carried out the MO infection experiment.The ELISA method and qRT-PCR method were used to analyze the influence of genes such as cytokines and TRAF6 in the NF-?B signaling pathway.Results:(1)After combined bioinformatics analysis,real-time fluorescence quantitative validation of miRNAs such as oar-miR-10 b,oar-miR-509-5p,oar-miR-432 was carried out,which confirmed the reliability of high-throughput sequencing database.Target gene prediction of miRNAs was carried out.Transcriptional databases of combined analysis showed that TRAF6 and STAT2 were differentially expressed genes.GO enrichment and KEGG analysis showed that differentially expressed miRNAs might be possible.It plays a role in inflammation.(2)According to the screening results,TRAF6 may be the target gene of miR-509-5p.The luciferase activity of wild-type plasmid of TRAF6 gene was significantly decreased after transfection with miR-509-5p mimics,but the mutant did not change significantly.This indicates that there is a targeting relationship between oar-miR-509-5p and TRAF6 gene.(3)Overexpression of miRNAs in respiratory epithelial cells significantly down-regulated the expression of TRAF6 and up-regulated the expression of TRAF6 after inhibiting the expression of miRNAs.Overexpression of miR-509-5p significantly increased the expression of TLR4 and IRAK4 in the NF-kappa B pathway.TAK1 gene was significantly down-regulated,significantly down-regulated the expression of NF-?B.Inhibition significantly increased the expression of NF-?B and significantly increased the expression of TAK1(4)PI staining showed that MBL protein had a protective effect on MO infected cells.The expression of cytokines IL-12,TNF-?,IL-6 and IL-1? was inhibited by over-expression of miR-509-5p in respiratory mucosal epithelial cells infected with MO.miR-509-5p may target TRAF6 and affect the expression of downstream genes.Conclusions:(1)Differential microRNAs and differentially expressed genes related to MBL resistance to Mycoplasma pneumonia in sheep were successfully screened.(2)It is proved that TRAF6 gene is the direct target gene of miR-509-5p.(3)It has been proved that miR-509-5p may target TRAF6 and affect the expression of downstream genes and cytokines in the NF-kappa B pathway.miR-509-5p may play a role in anti-mycoplasma sheep pneumonia.