Construction and Analysis for Dysregulated lncRNAs and mRNAs in Lipopolysaccharide-Induced Porcine Peripheral Blood Mononuclear Cells

Background: Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria induces an intense inammatory response in pigs. Long noncoding RNAs (lncRNAs) are emerging as key regulators in inammation and immunity. However, their functions and proles in LPS-induced inammation in pigs are largely unknown. In this study, we proled global lncRNA and mRNA expression changes in porcine peripheral blood mononuclear cells (PBMCs) treated with lipopolysaccharide (LPS) using lncRNA-seq technique. Result: Totally 43 differentially expressed (DE) lncRNAs and 1082 DE mRNAs were identied in the porcine PBMCs after LPS stimulation. Functional enrichment analysis on DE mRNAs indicated that these genes were involved in inammation-related signaling pathways, including cytokine-cytokine receptor interaction, TNF, NF-κB, Jak-STAT and TLR signaling pathways. Additionally, co-expression network and function analysis identied the potential lncRNAs related to inammatory response and immune response. The expression of eight lncRNAs (ENSSSCT00000045208, ENSSSCT00000051636, ENSSSCT00000049770, ENSSSCT00000050966, ENSSSCT00000047491, ENSSSCT00000049750, ENSSSCT00000054262 and ENSSSCT00000044651) was validated in the LPS- treated and non-treated PBMCs by quantitative real-time PCR (qRT-PCR). In LPS-challenged piglets, we identied that three lncRNAs (ENSSSCT00000051636, ENSSSCT00000049770, and ENSSSCT00000047491) were signicantly up-regulated in liver, spleen and jejunum tissues after LPS challenge, which revealed that these lncRNAs might be important regulators for inammation . Conclusion: This study provides the rst lncRNA and mRNA transcriptomic landscape of LPS-mediated

LPS-stimulated cells, and aggravates LPS-induced injury possibly via sponging miR-34a [7]. LncRNA-MALAT1 is induced by IL-6 in LPS-treated cardiomyocytes and its overexpression can enhance TNFexpression via activation of SAA3 [8]. The LPS-induced lncRNA Mirt2 functions as a repressor of in ammation through inhibition of TRAF6 oligomerization and auto-ubiquitination [9]. LncRNA GAS5 reverses LPS-induced in ammatory injury in ATDC5 chondrocytes by inhibiting the NF-κB and Notch signaling pathways [10]. However, the de nition of functional lncRNA in pigs are still limited, partly due to their low sequence conservation and lack of identi ed shared properties across species [11].
Considering the role of lncRNAs in the in ammatory response, the present study was designed to discover and explore lncRNA expression pro le of porcine PBMCs in response to LPS. To date, there has been no systematic attempt to identify the lncRNAs whose expression is changed after the induction of the innate immune response in pigs. This analysis of lncRNA expression changes in the porcine PBMCs after LPS stimulation would contribute to the current knowledge of lncRNA functions in gram-negative bacterial infection disease pathogenesis in pigs.
Quantitative RT-PCR Total RNA was isolated from cell and tissues samples using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA synthesis and quantitative real-time PCR (qRT-PCR) were carried out as previously descried [13]. Expression of mRNA and lncRNA was analyzed using the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was used as an internal normalization control. All data were analyzed using the 2 −∆∆CT method [14]. Sequences of speci c primers are shown in Additional le 1.

Cytokine TNF-measurement
The concentration of TNF-in the supernatants of PBMCs were measured using commercially available porcine ELISA kits (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instruction.
High-throughput sequencing RNA quality was examined by gel electrophoresis and only paired RNA with high quality was used for lncRNA-sEq. LncRNA-seq libraries were prepared according to the manufacturer's instructions and then applied to sequencing on Illumina HiSeq 3000 in Shanghai Genergy Co. Ltd (Shanghai, China). The original reads were harvested from the Illumina HiSeq sequencer. 3' adaptor-trimming and low-quality removal was performed with Trim Galore software (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), after which the resulting clean reads were used for lncRNA analysis. Clean reads were aligned to the pig reference genome (Sscrofa11.1) using STAR software (https://github.com/alexdobin/STAR).

LncRNA and mRNA differential expression analysis
To determine lncRNA and mRNA differential expression between the LPS-stimulated and unstimulated groups, the expression of each transcript was normalized to the total number of reads in the samples using the following formula: FPKM = total fragments / mapped reads (millions) * exon length (KB). The fold change in lncRNA or mRNA reads was presented as log2 transformation using the following formula: Fold change = log2 (LPS/Control). A adjust P-value less than 0.05 is considered signi cant.

Functional enrichment analysis
GO enrichment and KEGG analysis of DE mRNA was performed with DAVID 6.8 database (https://david.ncifcrf.gov/). Protein-protein interaction network between DE mRNA were analyzed by Ingenuity Pathway Analysis (IPA).

Target gene prediction and co-expression network construction
The predicted potential target genes whose loci were within a 10-kb window upstream or downstream of the lncRNA was considered cis-regulated genes. To determine the trans-regulated genes of the DE lncRNAs, the lncRNA and mRNA co-expression analysis were performed using Pearson correlation coe cient (PCC) method. The Pearson correlation coe cient was ≥ 0.9. The common genes between the potential targets of DE lncRNAs and DE mRNAs was analyzed using Venn analysis.
The network of coding-noncoding coexpressed genes was constructed with the biological functions to recognize the novel and signi cant lncRNA. Correlations between lncRNAs and their corresponding mRNAs were calculated with Pearson's correlation (|correlation|≥0.9) were used to draw the coexpression network through Cytoscape v 3.7.1.

Statistical analyses
The data are shown as means ± S.D. Differences were tested using ANOVA and the Student's paired t test. The level of signi cance was set at P < 0.05 for all data analysis.

Results
The expression of TNF-α, IL-1β and IL-6 in LPS induced PBMCs In order to identify principal LPS-responsive lncRNAs, PBMCs isolated from the whole blood of the three healthy pigs were stimulated with LPS for 4, 8, 12 and 24 h to induce in ammatory response. The mRNA of TNF-increased signi cantly in porcine PMBCs within 12 h and peaked at 4 h after LPS stimulation ( Fig. 1a). IL-6 and IL-1β were signi cantly increased at each time point and peaked at 8 h and 4 h, respectively ( Fig. 1b-c). We also applied ELISA to determine TNF-protein level in the cell culture supernatant. The concentration of TNF-was obviously increased by LPS stimulation compared to the control at each time point (Fig. 1d), and it was undetected in controls at 12 and 24 h. These results suggested an acute in ammation was induced by LPS stimulation in PBMCs. Then, the RNA samples isolated from PBMCs treated for 8 hours were used for further high-throughput sequencing.

Basic characteristics of lncRNAs in the PBMCs
The basic characteristics of all DE lncRNAs and DE mRNAs in PBMCs, which are widely distributed in all chromosomes except Y chromosome, are shown in the Circos plot (Fig. 3A). Next, we classi ed the PBMC lncRNAs into ve categories according to the genomic loci of their neighbouring genes (Fig. 3B). Although 10% of lncRNAs were not successfully categorized, the well-annotated lncRNAs were classifed into the following categories: intergenic (22%), antisense (9%), intronic (57%) and bidirectional (2%), and sense (0%).

Function analysis of DE mRNAs
The functional enrichment analysis of the 636 signi cantly up-regulated genes was performed. Our GO analysis included three parts: biological process (BP), cellular component (CC) and molecular function (MF). The top 15 GO enrichment for biological process for the up-regulated mRNAs was illustrated in Fig. 4a, including in ammatory response, immune response, positive regulation of in ammatory response and chemokine-mediated signaling pathway. The signi cantly enriched GO terms for cellular component and molecular function were identi ed, such as extracellular space, external side of plasma membrane, integral component of plasma membrane, cytoplasm, I-κB/NF-κB complex, cytokine activity, chemokine activity and growth factor activity.
The 446 signi cantly down-regulated genes were also selected to carry out the functional enrichment analysis. Twenty four GO terms, including protein localization to plasma membrane, regulation of Gprotein coupled receptor protein signaling pathway, cell surface, cell-cell junction, and kinase activity, were shown in Fig. 4b.
Meanwhile, KEGG results form the signi cantly up-and down-regulated genes indicated that the top 30 signi cantly signaling pathways were enriched (Fig. 4c), such as cytokine-cytokine receptor interaction, TNF signaling pathway, NF-κB signaling pathway, Jak-STAT signaling pathway, Chemokine signaling pathway and Toll-like receptor signaling pathway.
In addition, the interaction network between proteins was produced using IPA. The in ammatory immune network was shown in Fig. 4d. Seven candidate genes (NTRK1, S100A8, S100A9, TNIP1, TNFAIP3, TAX1BP1 and NOD2) were screened out as Hub genes.
Function analysis of DE lncRNA and the lncRNA-mRNA co-expression network A total of 1053 potential target genes of DE lncRNAs were selected to carry out functional enrichment analysis. Our results showed that the top 20 GO enrichment for biological process focused on immune response, in ammatory response, neutrophil chemotaxis, positive regulation of in ammatory response, chemokine-mediated signaling pathway, positive regulation of ERK1 and ERK2 cascade, lymphocyte chemotaxis, positive regulation of NF-κB import into nucleus, positive regulation of IL-6 production, necroptotic signaling pathway, monocyte chemotaxis, cell chemotaxis, positive regulation of NF-kappaB transcription factor activity, negative regulation of IL-10 production and lipopolysaccharide-mediated signaling pathway, etc. (Fig. 5a). Meanwhile, the KEGG results from these target genes of DE lncRNAs were mainly involved in immune response (Fig. 5b).
To identify the key lncRNAs related to the regulation of in ammatory response and immune response, 54 DE mRNAs associated with these two biological processes and 31 DE lncRNAs targeting them were chosen to build the mRNA-lncRNA co-expression network. The co-expression network comprised 1241 connections and each lncRNA might correlated with multiple mRNAs (Fig. 5c and Additional le 8). More importantly, a total of 29 lncRNAs were found to be co-expressed with chemokines (CCL2, CCL3L1,  CCL11, CCL17, CCL20, CCL22, CXCL2, CXCL8, CXCL10 and CXCL13) and cytokines (IL-1A, IL-6, IL-7, IL-10,  IL-12B, IL-13, IL-18, IL-19, IL-20, IL23A, IL-27). While, a total of 30 lncRNAs were shown to be co-expressed with other in ammatory-related genes such as Toll-like receptors (TLR2 and TLR3), the Rel/NF-kB family members (REL, RELB and NFKB2), and NFKBIZ encoding the NF-κB inhibitor. Three lncRNAs (ENSSSCT00000047491, ENSSSCT00000049770 and ENSSSCT00000051636) expressions were increased more than 2.5-fold in LPS-treated PBMCs at 4 h. Therefore, we further identi ed the expression changes of the three lncRNAs in various tissues of the piglets challenged with LPS at different time points.
Expression of lncRNAs (ENSSSCT00000047491, ENSSSCT00000049770 and ENSSSCT00000051636) in various tissues of piglets challenge with LPS As shown in Fig. 7a, in the liver tissue, lncRNA ENSSSCT00000047491 were dramatically up-regulated at least 4-fold by LPS from 2 h to 24 h, while the upregulation (> 9-fold) of lncRNA ENSSSCT00000051636 was observed after LPS challenge for 8 hours. LncRNA ENSSSCT00000049770 was dramatically (4 to 12-fold) up-regulated within 8 h but it was down-regulated at 24 h.
As shown in Fig. 7b, in the jejunum tissue, lncRNA ENSSSCT00000049770 was increased signi cantly at least 1.9-fold from 1 h until 24 h after LPS challenge, while the expression of lncRNA ENSSSCT00000051636 was signi cantly increased by > 2-fold from 1 h to 4 h. The expression of lncRNA ENSSSCT00000047491 was signi cantly up-regulated by LPS challenge at 4, 12 and 24 h.
As shown in Fig. 7c, in the spleen tissue, the expression of the three lncRNAs was signi cantly increased within 8 h and reached the peak at 2 h, but their expression was decreased at 24 h.
As shown in Fig. 7d, in the thymus tissue, the expression of the three lncRNAs showed no signi cant difference.
Tissue expression pattern analysis of lncRNA ENSSSCT00000047491, ENSSSCT00000049770 and ENSSSCT00000051636 We further detected the differences of the three lncRNAs (ENSSSCT00000049770, ENSSSCT00000051636 and ENSSSCT00000047491) expression levels in piglets varieties tissues by qRT-PCR. The results showed that the three lncRNAs were expressed in all of the ten tissues: skeletal muscle, heart, liver, spleen, lung, kidney, jejunum, stomach, brain and thymus. We also found that the expression levels of lncRNA ENSSSCT00000047491 and ENSSSCT00000051636 were higher in liver and spleen than in other tissues, while the expression level of lncRNA ENSSSCT00000049770 were higher in spleen, lung and jejunum than in other tissues (Fig. 8).

Discussion
Accumulating evidence has indicated that lncRNAs play roles in immune/in ammatory processes [15,16]. respectively [17]. However, to date there has been no systematic attempt to identify LPS-associated lncRNAs in porcine PBMCs.
In this study, we provides the rst lncRNA and mRNA transcriptomic landscape of LPS-mediated changes in porcine PBMCs. Using a sequencing approach, we obtained 1074 lncRNAs and 27430 mRNAs in porcine PBMCs. Of the 1074 lncRNAs, we identi ed 31 up-regulated and 12 down-regulated lncRNAs in LPS-treated PBMCs compared with the control. Of the 27430 mRNAs, 636 mRNAs were up-regulated and 446 mRNAs were down-regulated by LPS stimulation. Similarly to the results described by Zhang et al. [17] in human PBMCs, we found more mRNAs than lncRNAs were dysregulated in response to LPS.
Moreover, more transcripts of the dysregulated mRNA or lncRNAs were observed to be up-regulated after LPS stimulation in human and porcine PBMCs. In our recent study, we had investigated the changes in miRNA expression in porcine PBMCs after in vitro stimulation with LPS and identi ed only 15 DE miRNA in response to LPS by using small RNA sequencing [12]. Therefore, we thought LPS stimulation might have more in uence on protein coding RNA more than non-coding RNA in PBMCs.
It was well recognized that a series of genes involved in LPS-induced in ammation [18][19][20]. In this study, the DE mRNAs functional enrichment results showed that these genes were related to some biological processes, including in ammatory response, immune response, cytokine activity, chemokine activity, cytokine-cytokine receptor interaction, TNF signal pathway, NF-κB signal pathway, JAK-STAT signal pathway, NOD-like receptor signal pathway and TLR signal pathway, which were closely associated with LPS-induced in ammation [21][22][23][24][25]. In addition, though the IPA network analysis, we identi ed some genes might play key roles in LPS-induced in ammation, including NTRK1, S100A8, S100A9, TNFAIP3, TNIP1, TAX1BP1 and NOD2. As a high-a nity receptor for nerve growth factor (NGF), NTRK1 is expressed on various structural and hematopoietic cells including basophils and eosinophils [26,27]. Rochman et al. [28] demonstrated IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and this process likely contributes to allergic in ammation. Calcium-binding proteins S100A8 and S100A9 have been identi ed as important DAMPs and recognized by TLR4 on monocytes, which function as innate ampli er of infection, autoimmunity, and cancer [29,30]. The TNFAIP3 gene, encoding the ubiquitin-modifying enzyme A20, that restricts NF-κB-dependent signaling and prevents in ammation via its deubiquitinase activity [31]. TNIP1 is increasingly being recognized as a key anti-in ammatory protein by negatively regulating TANK-binding kinase 1 (TBK1), receptor-interacting serine/threonine kinase 1 (RIP1 or RIPK1), and interleukin-1 receptor-associated kinase 1 (IRAK1) [32][33][34][35]. TAX1BP1 is a negative regulator of NF-κB activation induced by TNF-α and IL-1β [36]. It inhibits RIP1 and TRAF6 polyubiquitination and recruits A20 to these molecules in order to in uence NF-κB activation [37]. NOD2 is a macrophage-speci c protein containing two CARD domains and can directly bind bacterial lipopolysaccharide and subsequently act as an activator of NF-κB via the association of the CARD domains with Rip2/RICK/CARDIAK [38].
LncRNAs can be categorized into ve broad subcategories: antisense, sense, intergenic, intronic, and bidirectional [39]. Approximately half of lncRNAs in porcine PBMCs belonged to the intronic subcategory, which describes lncRNAs that are located within protein-coding genes and could regulate functional gene expression. Based on their mode of action on gene expression, lncRNAs can be classi ed as either cis-or trans-acting. Cis-acting lncRNAs affect the expression of genes located near their site of transcription on the same chromosome. Trans-acting lncRNAs can control gene expression at independent loci on other chromosomes [40]. Then, we predicted the cis and trans potential targets of DE lncRNAs and compared these with our mRNAs sequencing results. The DE lncRNA-associated DE mRNAs were further analyzed for GO category and KEGG pathway annotation to investigate the potential regulatory roles of LPSmediated DE lncRNAs. Bio-informatics analysis of DE lncRNAs target genes showed that these genes played important roles in immune response, in ammation, positive regulation of ERK1 and ERK2 cascade, positive regulation of NF-κB import into nucleus, positive regulation of IL-6 production and immune cell chemotaxis. ERK1 and ERK2 are reported to be required for LPS-induced production of cytokines and chemokines by macrophages [41]. KEGG analysis also indicated that the DE lncRNAs were predominantly associated with the regulation of multiple in ammatory associated genes. In addition, the lncRNA-mRNA co-expression analysis showed revealed that 29 DE lncRNAs targeted CCL and CXCL chemokines, indicating that these lncRNAs might participate in immune cell chemotaxis. The coexpressed network showed that multiple lncRNAs could interact with NFKBIA, which conjugated with NF-κB resulting in its cytoplasmic sequestration and inhibition of transcriptional activation [42]. Consequently, our results provide new evidence that lncRNAs are involved in LPS-indcued in ammation in porcine PBMCs.
Subsequently, 9 DE lncRNAs were selected for validation through qRT-PCR. For the nine lncRNAs, 8 selected lncRNs were validated to be signi cantly up-regulated by LPS stimulation at different times. The three lncRNAs (ENSSSCT0000004749, ENSSSCT00000049770 and ENSSSCT00000051636) was increased more than 2.5-fold after LPS stimulation in porcine PBMCs. Then, we further con rmed the expression changes of the three lncRNAs in liver, spleen, jejunum and thymus of the piglets challenged with LPS at different times (0, 1, 2, 4, 8, 12 and 24 h). The previous studies demonstrated LPS challenge can induce sever in ammation in the piglet model, causing liver injury, intestinal damage and histological changes of spleen [43][44][45][46][47]. Although the three lncRNAs displayed different tissue expression patterns in the piglets, they all shown abundant expression in liver, spleen, jejunum and thymus. In liver tissues, lncRNA ENSSSCT00000047491 expression increased approximately 4-to 33-fold, lncRNA ENSSSCT00000049770 increased 4-to 12-fold and lncRNA ENSSSCT00000051636 increased 9-to 64fold in response to LPS challenge. In the spleen and jejunum, LPS challenge induced an increase in these lncRNAs expression with a maximum response of 4-fold increase. However, in the thymus, LPS challenge had no effect on the expressions of the three lncRNAs. These results indicated the three lncRNAs might have different function in different tissues. We predicated only FLYWCH2 gene was the cis-target of lncRNA ENSSSCT00000049770 and also co-expressed with this lncRNA. However, there is very little speci c information on the expression and function of FLYWCH2 gene in in ammation.

Conclusions
In the current study, we have provided the changes of lncRNA expression in porcine PBMCs after LPS stimulation, which provided a novel foundation for improving our understanding of the association between PBMC lncRNA homeostasis and in ammatory response in pigs. Further investigations are still required to evaluate the biological functions of these identi ed lncRNAs and these signaling pathways with regard to their roles in immunity and disease.         The expression pattern of the three lncRNAs (ENSSSCT0000004749, ENSSSCT00000049770 and ENSSSCT00000051636) in porcine various tissues. The data represent the mean ± SD. N=2.