JIA-2019-11

2593 BI Yu-lin et al. Journal of Integrative Agriculture 2019, 18(11): 2589–2597 To predict and certify the relationship between the candidate lncRNAs and their target genes, gene sequence structure was analyzed on e-Ensembl websites. Candidate L11530 (6.4 kb in length) was 559 bp distant from the target gene, NOD1 , located in the upstream transcription start area, and up-regulated NOD1 gene expression via cis -regulation (Fig. 2-C). Similarly, the candidate L09863 (2.4 kb in length), which was 3.47 kb distant from the target gene, TRAF5 , located upstream of the transcription start area and up- regulated the expression of TRAF5 , also by cis -regulation in the same direction (Fig. 2-D). These results reinforced the creditability of the data in this study. 3.3. Expression of two candidate lncRNAs and their target genes The expression levels of L11530, L09863, and the target genes NOD1 and TRAF5 were detected via Q-PCR in vitro to validate the relationship between the identified lncRNAs and target genes. The results showed that the expression levels of L11530 and NOD1 had the same trend, being significantly up-regulated ( P <0.01 and P <0.01, respectively), and the expression levels of L09863 and TRAF5 were significantly down-regulated ( P <0.05 or P <0.01) in lymphocytes infected by REV (Fig. 3-A). Moreover, lymphocytes in blood were isolated from chickens with infection by REV or not, and directly used to further validate the above results in vitro . It was found that the expression levels of L11530 and NOD1 were significantly up-regulated ( P <0.01 and P <0.01, respectively), but the expressions of L09863 and TRAF5 were significantly down-regulated ( P <0.05 and P <0.05, respectively) in lymphocytes of chickens with REV infection compared to those uninfected by REV (Fig. 3-B). These results demonstrated the consistency of the expression levels of lncRNAs (L11530, L09863) and their corresponding target genes ( NOD1 and TRAF5 ) in vitro and in vivo . To further verify the relationship between two lncRNAs and their candidate target genes, the interfered results by the specific siRNAs showed the L11530 in REV-infected lymphocytes or L09863 in uninfected lymphocytes Fig. 1 The effectiveness verification of reticuloendotheliosis virus (REV) infection and data by RNA-seq. Lymphocytes were infected by REV for 36 h in vitro and the uninfected cells were the control. A, DNA level of long terminal repeat (LTR). M, DL2000; +, positive control; –, negative control; I, the sample of cells with infected by REV. B, the level of IL-8 and IL-18 in culture medium. C and D, the volcano and csScatter analyses based on 134 screened differentially expressed lncRNAs. E, the correlation analysis by Q-PCR and RNA-seq ( r =0.9275, P <0.01). Data are mean±SEM ( n =3). 0 0.1 0.2 0.3 0.4 IL8 IL18 Immune factor level (ng mL –1 ) Control REV * * M + – REV 288 bp y =0.88x+0.1652 r =0.9275 –6 –4 –2 0 2 4 6 –6 –4 –2 0 2 4 6 C B A D E 4 2 0 4 2 0 0 2 4 0 2 4 4 3 2 1 0 4 3 2 1 0 –10 –5 0 5 10 log 2 (fold change) log 10 ( P -value) log 10 FPKM log 10 FPKM –10 –5 0 5 10 c0 c5 c0 c5 Significant No Yes c5 c0 c5 c0 Q-PCR RNA-seq