Effect Of MiR-200b-3p On Chicken Antigen-presenting Pathway

MicroRNAs are a group of non-coding single-stranded RNA molecules with a length of about 22 nucleotides.They are widely present in eukaryotic organisms and participate in the regulation of a variety of life processes.MiRNAs play a widely gene regulation functions in the growth,development,metabolism and other physiological processes of chickens.They also play an important role in the regulation of viral infection,apoptosis,monocyte/macrophage differentiation,and immune cell development and function.In this study,small RNA-seq was performed on spleen and bursa of SPF chickens in unimmunized and immunized groups.Through analysis,we filtered the miRNAs associated with antigen presentation and used miR-200b-3p as candidate for the study of chicken antigen presentation.On the one hand,we studied the effect of miR-200b-3p on antigen presentation.On the other hand,the molecular mechanism of miR-200b-3p and its target genes on antigen presentation was studied.The main results of the paper were showed as follows1.Small RNA-seq results showed that there were 36 and 43 miRNAs significantly differently expressed(P<0.05)in bursa and spleen after immunization.GO function enrichment analysis showed that potential target genes of candidate miRNAs were significantly enriched(P<0.05)in protein binding,transcription factor binding,metabolic processes,T cell activation,immune system processes,immune synapses.KEGG pathway analysis showed that potential target genes of candidate miRNAs were significantly enriched(P<0.05)in oocyte meiosis,mTOR signaling pathway,TGF-? signaling pathway and cell cycle pathway.Combined with the above analysis,it was speculated that miR-200b-3p and its target genes MMD and DUSP1 may participate in antigen presentation.2.HD11 cells overexpressed and inhibited with miR-200b-3p were detected by qRT-PCR and Flow Cytometry.The results showed that miR-200b-3p could promote the expression of CD80,CD86 and other antigen-presenting genes in HD11 cells,and also could promote the expression of MHC-II on the surface of HD11 cells.3.Using TargetScan and miRDB to predict target genes of gga-miR-200b-3p,combined with previous studies and literature reports,we selected DUSP1 and MMD as candidate target genes.Dual luciferase reporter assays determined that DUSP1 and MMD were target genes for miR-200b-3p,and qRT-PCR indicated that miR-200b-3p could inhibit the expression of DUSP1 and MMD.4.In HD11 cells,overexpression of MMD could inhibit the expression of IL6,MHC-II and other antigen-presenting genes.