JIA-2019-11

2594 BI Yu-lin et al. Journal of Integrative Agriculture 2019, 18(11): 2589–2597 were significantly down-regulated ( P <0.01 and P <0.01, respectively), accompanied by significant down-regulation of their corresponding target genes NOD1 and TRAF5 ( P <0.01 and P <0.01, respectively) (Fig. 3-C). In the above two treatments, the secretion level of IL-8 and IL-18 were significantly reduced after L11530 interfering in REV-infected lymphocytes, and significantly increased ( P <0.05) after L09863 interfering in uninfected lymphocytes (Fig. 3-D). 4. Discussion REV infection of lymphocytes or reticuloendothelial cells leads to immuno-suppression in chickens (Witter et al . 1979, 1981). The pathogenesis of REV has been established (Li et al . 2016; Xue et al . 2017), but the mechanism of immune response to REV in chickens is still poorly understood. LncRNAs are important in regulating the expression of genes (Alvarez-Dominguez and Lodish 2017; Liu et al . 2017). To provide further clarification of the mechanism of REV resistance in lymphocytes from chicken blood, we presently screened new lncRNAs and their target genes involved in immune regulation. As the terminal expression products in the NOD1 pathway, IL-8 and IL-18 were used to estimate the effect of REV infection of lymphocytes, since their expression levels decrease during tumor development (Huang et al . 2015; Li et al . 2017) and their secretion levels would reduce for REV infection (Bi et al . 2018). After lymphocytes were infected by REV (10 5 TCID 50 /0.1 mL) for 36 h, except for the finding of very high level of LTR in REV-infected lymphocytes, both the mRNA and protein levels of IL-8 and IL-18 were significantly down-regulated compared with the control cells. The results provided evidence of the successful infection of lymphocytes by REV. Similarly treated samples were used in the subsequent experiments. RNA-seq revealed that 134 lncRNAs were differentially expressed in lymphocytes with or without REV infection. The csScatter and volcano analyses indicated the accuracy of lncRNAs data from RNA-sequencing. The expressions of ten lncRNAs were additionally detected by Q-PCR. The correlation efficiency of Q-PCR and RNA-seq data ( r =0.9275) supported the accuracy of the sequence data. The 14 enriched pathways were screened via KEGG analysis based on differently expressed mRNAs, including the Salmonella infection signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway, which are important in the regulation of the immune response (Man et al . 2013; Xiao et al . 2015; Wang et al . Fig. 2 The analysis of two long non-coding RNAs (lncRNAs) (L11530 and L09863) and their target genes. A, the 14 enriched pathways in lymphocytes with or without reticuloendotheliosis virus (REV) infection. B and C, analysis of gene sequence structure and regulation relationship of lncRNAs (L11530 and L09863) and their target genes ( NOD1 and TRAF5 ). D, the prediction on target genes of L11530 and L09863 based on the screened enriched pathways. TRAF5 16.1 kb LncRNA 2.4 kb Cis -action Chr. 3: 22288300 22290742 22293470 22309604 NOD1 23.1 kb LncRNA 6.4 kb Cis -action Chr. 2: 41812538 41818975 41812538 41818975 ENSGALG00000011530 ENSGALG00000009863 D C A B