Effects Of Probiotics On Expression Of MiRNAsin Salmonella-challenged Broiler Chicks

The microRNAs(miRNAs)have been shown to play important roles in the development of the immune system and in regulation of host inflammation responses.Probiotics can effectively alleviate the inflammation caused by Salmonella in chickens.However,whether and how miRNAs are involved in modulation of the inflammation response in the gut of chickens have not been reported.In this study,the impact of a probiotics,Lactobacillus plantarum Z01(LPZ01),was investigated on the cecal mi RNAs and cytokine secretions in Salmonella Typhimurium(S.Typhimurium)-infected chickens at the age of 3 days.1.Newly hatched chicks were assigned to four groups(1): NC(basal diet)(2): S(basal diet + S.Typhimurium challenged)(3): SP(basal diet + S.Typhimurium challenged + LPZ01)(4): P(basal diet + LPZ01).Expression of 14 miRNAs was significantly affected by the presence of S.Typhimurium and/or Lactobacillus plantarum Z01.Five differential expressed miRNAs(gga-miR-215-5p,gga-mi R-3525,gga-miR-193a-5p,gga-miR-122-5p,and gga-miR-375)were selected for confirmation by the RT-PCR.2.Predicted target genes of differentially expressed miRNAs were enriched in regulation of cAMP-dependent protein kinase activity,stress-activated MAPK cascade,immune system development and regulation of immune system process as well as in immune related pathways such as MAPK and Wnt signaling pathways.3.In comparison with the S group,chicks in the SP group reduced the number of S.Typhimurium and had lower levels of interferon-? and lipopolysaccharide-induced tumor necrosis factor alpha factor(LITAF)in ceca post challenge.4.The relationship between changes of miRNAs and changes of cytokines was explored.No immune related target genes were found for gga-mi R-2954.Using vanin 1(VNN1),LITAF,IFN-? and IL-6 as the main immune target genes,the remaining mi RNAs were involved directly or indirectly in regulation of cytokine production,thus affecting the host immune responses to the bacteria infection.In particular,miR-3525 and miR-193a-5p were found to indirectly target IL-6 and IFN-?.Finally,119 novel miRNAs were identified in 36 libraries totally.Identification of novel miRNAs significantly expanded the repertoire of chicken miRNAs and provided the basis for understanding the function of miRNAs in thehost.Our results suggest that the probiotics reduce the inflammation of the S.Typhimurium infection in neonatal broiler chicks,at least partially,through regulation of miRNAs expression.5.Among the differential expression miRNAs,the mi R-193a-5p was selected for the down-stream target genes search.Eight immuene related target genes(TRAF3,C1QTNF2,IL1RAPL1,MMP16,TRAT1,CREB3L2,IFNA7,TNFAIP1)of miR-193a-5p were predicted by using the online database miRDB,TargetScan and miRecords and selected.Then TRAF3 was validated by an in vitro luciferase reporter assay.Overall,the current study suggests that miR-193a-5p is expressed in chicken ceca and tagets TRAF3,which is likely to be involved in the regulation of host immune reaction and inflammation regulation.Overall,the current study suggests that expression of 14 miRNAs and cytokines(LITAF,IFN-? and IL-6)were significantly affected by the presence of S.Typhimurium and/or Lactobacillus plantarum Z01.The differentially expressed miRNAs were involved directly or indirectly in regulation of cytokine production,thus affecting the host immune responses to the bacteria infection.Among the differential expression miRNAs,the miR-193a-5p tagets TRAF3 in Caco-2 cells and chicken primary intestinal epithelium cell.The study has important theoretical and application value for understanding the interaction of intestinal microorganisms and host and the regulation function of intestinal microorganisms to host non-coding RNA.