JIA-2019-11

2595 BI Yu-lin et al. Journal of Integrative Agriculture 2019, 18(11): 2589–2597 2017). Two genes of the NOD-like receptor pathway, NOD1 and TRAF5 , were predicted target genes of L11530 ( NOD1 ) or L09863 ( TRAF5 ) in the analysis of the target genes of 134 lncRNAs. Considering the importance of NOD1 and TRAF5 in immune regulation (Buchta and Bishop 2013; Bi et al . 2017; Keestra-Gounder and Tsolis 2017), these results indicate that both two lncRNAs might participate in regulating the immune response in blood lymphocytes after REV infection. The lncRNAs regulate the expression of target genes with various ways (Wang et al . 2008; Sharma et al . 2011). Gene sequence structure analysis was performed using e-Ensembl sites to interpret the relationship between the candidate lncRNAs and their target genes. The results revealed two candidates: L11530 (6.4 kb) and L09863 (2.4 kb). These were located in the upstream transcription start area of the corresponding target gene. They positively regulated the expression of NOD1 and TRAF5 genes by cis -regulation. Finally, using the blood lymphocytes treated by REV in vitro and in vivo , the expression of NOD1 , TRAF5 , L09863, and L11530 were detected by Q-PCR. NOD1 was significantly up-regulated and TRAF5 was significantly down-regulated. TRAF5 can negatively regulate immune action (Buchta and Bishop 2013), while NOD1 can improve the immune response (Keestra-Gounder and Tsolis 2017). Further, L09863 and L11530 were respectively interfered, the expression levels of their target genes NOD1 or TRAF5 were significantly down-regulated, accompanied by the change of IL-8 and IL-18 secretions in lymphocytes. The present results clarify the importance of the NOD-like receptor pathway in immune regulation during REV infection. As expected, the expression of L11530 and L09863 significantly up-regulated and down-regulated their target genes, respectively, indicating consistency in expression levels of lncRNA and its target gene ( NOD1 or TRAF5 ) with the same change in vivo and in vitro . These data also strongly support the view that lncRNAs (L11530 and L09863) are involved in immune action during REV infection by regulating the expression level of the corresponding target genes ( NOD1 and TRAF5 ). Considering the consistency of all the above results on the levels of IL8 and IL18, the enriched regulatory factors in NOD-MAPK-AP1 pathway and the expression levels of two lncRNAs and their target genes, the proposed molecular regulatory network activating NOD-like receptor pathway after REV infection is presented in Fig. 4. The present approach used RNA-seq to elucidate immune mechanisms in lymphocytes of chickens after REV infection. A more Fig. 3 The changes of the expressions of two candidate long non-coding RNAs (lncRNAs) and immune factors ( IL-8 and IL-18 ). A, B and C, the expression levels of L11530, L09863, and the target genes NOD1 and TRAF5 in lymphocytes with different treatments in vivo and in vitro by Q-PCR. D, the levels of IL-8 and IL-18 in culture medium after L11530 or L09863 were interfered in vitro . Data are mean±SE. n =10 ( in vivo ) and n =3 ( in vitro ). * ** 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 L11530 NOD1 L09863 TRAF5 Expression level of lncRNA and the target gene in vitro (fold change) Control- in vitro REV- in vitro ** ** ** A B C D 0 0.5 1.0 1.5 2.0 2.5 3.0 L11530 NOD1 L09863 TRAF5 Expression level of lncRNA and the target gene in vitro (fold change) Control- in vivo REV- in vivo ** * * 0 0.2 0.4 0.6 0.8 1.0 1.2 L11530 NOD1 L09863 TRAF5 REV- in vitro Control- in vitro Expression level of lncRNA and the target gene in vitro (fold change) si-NC siRNA ** ** ** ** 0 0.1 0.2 0.3 0.4 0.5 IL-8 IL-18 IL-8 IL-18 REV- in vitro Control- in vitro The levels of IL-8 and IL-18 in vitro (ng mL –1 ) si-NC siRNA ** ** ** *