JIA-2019-11

2592 BI Yu-lin et al. Journal of Integrative Agriculture 2019, 18(11): 2589–2597 2.7. siRNA of lncRNA and transfection Acco r d i ng t o t he da t abase sequences o f two lncRNAs (L11530, ENSGALG00000011530; L09863, ENSGALG00000009863), and three siRNAs (L11530, UCACACCCCUUAUCACACC; L09863, AGUUCGAGA CGUGCCAAGC; si-NC, CUCCUUAAGUGGCGCCGAA) were designed and customized for production by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Lymphocytes in 96-well plate or 100-mm dish were infected with or without REV in vitro for 12 h. And then, the siRNA of L11530 was transfected in lymphocytes with REV infection and the siRNA of L09863 in lymphocytes without REV infection, and their corresponding si-NC as the control. After the cells were cultured for another 36 h, the IL-8 level, the expression of two lncRNAs and their target genes were detected. 2.8. Enzyme-linked immunosorbent assay (ELISA) In accordance with the manufacturer’s instructions, the cell mediums were collected from different groups and used for the detection of IL-8 or IL-18 using specific ELISAKit (R&D, Systems, Minneapolis, MA, USA). 2.9. Real-time quantitative PCR (Q-PCR) The Q-PCR was performed to detect the expression levels of lncRNAand gene mRNA, their specific primers were listed in Table 1, and β-actin was used as a house-keeping gene. The cDNA was obtained by using a RevertAid First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Each 20 μL PCR mixture contained 10 μL of the 2× iQ™ SYBR Green Supermix, 50 ng cDNA, and 0.5 μL (10 mmol L –1 ) of each primer. Mixtures were incubated in an ABI7500 Real-Time PCR Detection System (Applied Biosystems, Carlsbad, CA, USA). Amelting curve was constructed to verify that only a single PCR product was amplified. Samples were assayed in triplicate with standard deviations of threshold cycle (C T ) values not exceeding 0.5 on a within-run basis. Correlation analysis for gene expression between two methods was performed. 2.10. Statistical analyses Statistical differences between groups were evaluated using the Student’s t -test. P <0.05 ( * ) or <0.01 ( ** ) was considered significant. Data were represented as the mean±SE. Data analyses were performed using SPSS Software version 20.0 (IBM Inc., New York, NY, USA) (Saeid et al . 2016). Each replicate was treated as an experimental unit. The gene sequence structure was analyzed on the e-Ensembl website (https://asia.ensembl.org/index.html) . 3. Results 3.1. Change of immune factor levels and differentially expressed lncRNAs in lymphocytes after REV infection After lymphocytes were infected in vitro for 36 h, the LTR of REV was detected at an extremely high level by PCR (Fig. 1-A), indicating that lymphocytes were successfully infected by REV. Immune factors IL-8 and IL-18 were used as other criteria of successful infection of lymphocytes by REV. After the infection by REV for 36 h, the levels of IL-8 and IL-18 were significantly decreased (both P <0.05) in lymphocytes for REV (Fig. 1-B). Screening revealed 134 lncRNAs (26 up-regulated and 108 down-regulated) were differentially expressed between lymphocytes uninfected and infected by REV (Appendix A). To validate the sequencing data, csScatter and volcano analyses of RNA-seq were performed. The results indicated the accuracy of data from RNA-seq (Fig. 1-C and D). Ten of the 134 screened lncRNAs (five up-regulated and five down- regulated) were randomly selected to detect their expression by Q-PCR and to validate the accuracy of data by RNA-seq, and the correlation coefficient of data by Q-PCR and RNA- seq was calculated as ( r =0.9275, P <0.01), confirming the accuracy of the data by RNA-sequencing (Fig. 1-E). 3.2. Characterization of two candidate lncRNAs related to immune in NOD-like receptor pathway based on differently expressed protein-coding genes Based on differently expressed protein-coding genes, the 14 enriched pathways were screened by KEGG analysis (Fig. 2-A), these pathways were involved in immune response (Salmonella infection signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway) and other pathways. The target genes of 134 differently expressed lncRNAs were also analyzed and listed in Appendix B. Two target genes related to the immune response were found: NOD1 (chr. 2: 41819534–41843672) and TRAF5 (chr. 3: 22293470–22309604). An in-depth assessment of the relationship between these target genes and the three aforementioned pathways related to the immune response revealed both NOD1 and TRAF5 in the NOD-like receptor signaling pathway (Fig. 2-B). Their corresponding lncRNAs were ENSGALG00000011530 (L11530, chr.2: 41812538–41818975), and ENSGALG00000009863 (L09863, chr. 3: 22288300–22290742), respectively. These results indicated that these two lncRNAs might participate in regulating the immune response in blood lymphocytes during REV infection.