4.1. lncRNAs Were Invoved in Immune-Ralated Pathways
Long non-coding RNAs, defined as transcripts with a length greater than 200 nucleotides and a lack of protein-coding ability, are a group of non-coding RNAs with the largest number of bases generated in the genome. LncRNAs regulate gene expression at transcriptional and post-transcriptional levels through a variety of mechanisms. They play a key role in biological processes and diseases such as cell differentiation, tissue and organ development, and cancer metastasis [
25]. A number of lncRNAs have been identified from humans, rats, mice, bovines, sheep, goats, and other species by high-throughput sequencing [
8]. For goats, data have also been reported on lncRNAs in skeletal muscle [
26], hair follicles [
27], mammary glands [
28], and other tissues, but no reports have been made on lncRNAs in the submandibular glands. Although the role of coding genes in immune cell function has been well described, the study of immune-related lncRNAs has just begun to emerge [
29]. LncRNAs are a key regulator of gene transcription during inflammatory responses. In the innate immune system, lncRNAs play a role by regulating the inflammatory process [
30]. In the acquired immune system, the function of
NFAT1 in T cells was regulated by lncRNA: NRON (ncRNA repressor of NFAT) [
31]. Maladjusted lncRNAs in human diseases such as inflammatory bowel disease, diabetes, allergies, asthma, and cancer have been found to be crucial for immune mechanisms. They are involved in cell differentiation, migration, and production of inflammatory mediators by regulating protein-protein interactions or by their assembly with RNA and DNA, and finally, they may affect all possible lncRNA biological effects through cis or trans interactions [
32].
In this study, a total of 4404 lncRNAs were identified from the SMGs of 1-month-old goat kids, 12-month-old unbred adolescent goats, and 24-month-old adult goats (three samples in each group) by high-throughput sequencing. The number of known lncRNA transcripts in the three groups accounted for 60.80%, 55.75%, and 51.61% of the total lncRNA transcripts, respectively. This indicates that the number of known lncRNAs obtained by goat SMGs accounts for more than half of the total number of lncRNAs. There were only 11 highly expressed lncRNAs, indicating that the expression of lncRNAs was generally low in different developmental stages of goat SMGs. The results of differential expression analysis indicated that the expression pattern of lncRNAs in the goat SMGs was similar between adolescent and adult goats, but the expression pattern of lncRNAs changed greatly from 1-month-old goat kids to adolescent goats, which may indicate that the expression of lncRNAs is time and tissue specific during the growth and development of goat SMGs.
To predict the function of lncRNAs, predictive analysis of the interaction mode of all DElncRNAs and enrichment analysis for targeted mRNAs with different modes were performed. The results showed that the targeted mRNAs with different modes were partially enriched in protein metabolism, lipid metabolism, and other related pathways, indicating that the expression of digestive enzymes in goat SMGs was regulated by lncRNAs. Significant enrichment results showed that the main mode of lncRNA regulation of digestive enzymes was cis-acting.
The top three pathways significantly enriched for cis-acting target genes, the NF-κβ signaling pathway, the T cell receptor signaling pathway, and the intestinal immune network producing IgA, are all immune-related pathways, and their target genes are all up-regulated, indicating that the lncRNAs are involved in the three pathways in SMGs by up-regulating target mRNAs through cis-acting mode. We noticed that about one-third of the functions of trans-acting lncRNA targeted mRNAs were immune-related biological processes, and most of the significantly enriched pathways were also immune-related pathways. Therefore, we predict that DElncRNAs in goat SMGs have a regulatory role in immune correlation, but the specific regulatory mechanism needs to be further studied. As core genes in the NF-κβ signaling pathway and the T cell receptor signaling pathway,
V-TCR were cis-regulated by TCONS_00013148 and trans-regulated by TCONS_00041162 and TCONS_00052910. As well as
TCRB, a core gene in the above two pathways, was trans-regulated by XR_001297267.2. TCR (T-cell antigen receptor) is a heterodimeric glycoprotein composed of two peptide chains, TCR-α and TCR-β. Each peptide chain is generated by genomic rearrangements of the variable, diverse, connecting, and constant regions segments, and TCR-β contains 55 different V regions [
33]. The results suggested that different types of lncRNAs may regulate different V regions of the TCR-β chain in the process of regulating
V-TCR. Fyn is a member of the Src family tyrosine kinases with a molecular weight of 59 kDa and has diverse biological functions [
34]. The study by Oers et al. implied a critical role for Lck and Fyn in T-cell development [
35]. The TCONS_00072725 cis-acting target gene
PKCƟ (encoded by PRKCQ) played a key role in regulating the differentiation and proliferation of T lymphocytes [
36], and its mutation can cause the occurrence of Crohn’s disease and other diseases [
37]. Prediction results have showed that TCONS_00029100 further affected the function of NKG2D through cis-binding to the target gene NKG2D ligand, LOC102185264. It can be seen that TCONS_00013148, TCONS_00041162, TCONS_00052910, TCONS_00072725, XR_001297267.2, and TCONS_00029100 cis/trans target mRNAs were related to the immune function of T cells. Moreover, these 6 lncRNAs were highly expressed in the SMGs of 1-month-old goat kids and lowly expressed in 12-month-old unbred adolescent goats and 24-month-old adult goats, which may be one of the reasons that the expression of immune-related genes was significantly down-regulated with the growth and development of goat SMGs.
Membrane-spanning 4-domain family gene (MS4A) is mainly expressed in lymphoid nuclei and blood, and plays a key role in regulating cell activation, growth, and development. Unlike other lncRNAs, TCONS_00078593, which trans-acted with target gene HSP90AA1 (which encode the heat shock protein Hsp90α), was significantly down-regulated in the SMGs of 1-month-old goat kids and up-regulated in 12-month-old unbred adolescent goats and 24-month-old adult goats. Previous studies have found that Hsp90α is associated with scrapie in goats, thus, TCONS_00078593 might be a biomarker for scrapie in goats.
Chemokines are essential for the tissue-specific migration and localization of immune cells in lymphoid organs. Without inflammation, the receptor CCR4 is predominantly expressed by regulatory T cells (T Reg) [
38]. By researching the CCL22-deficient mice, Moritz Rapp et al. found that the expression of
CCL22 by dendritic cells (DCS) promoted cell-to-cell contact and interaction with regulatory T cells (T Reg) through the CCR4 receptor, and CCL22-deficient mice had increased susceptibility to inflammatory diseases. Therefore, the CCL22-CCR4 axis is an important immune checkpoint to control T-cell immunity [
39]. Tyner et al. found that when mice lacking the chemokine CCL5 were infected by parainfluenza or human influenza virus, impaired immunity leads to viral clearance delaying, excessive airway inflammation, and even respiratory death [
40]. In this study, we found that XR_310184.3 and TCONS_00072236 trans-acted target genes
CCL22 and
CCR4. Therefore, the co-expression of these two lncRNAs may affect the immune function of T cells. Moreover, we found they were significantly down-regulated in 12-month-old unbred adolescent goats and 24-month-old adult goats, which may be another influencing factor for the down-regulation of immune-related genes in goat SMGs. Chemokine CCL5, after binding with receptor CCR5, can activate the PI3K-AKT and MEK-ERK signaling pathways to play an immune protective role. However, in goat SMGs, we found that the significantly upregulated TCONS_00020715 trans targeted gene
CCL5 was negatively correlated, which seems to indicate that the ability of goat SMGs to initiate related signaling pathways through CCL5 to cope with viral infection is weakened with age. Interleukin 7 (IL-7) and its receptor are formed by IL-7Rα (encoded by
IL-7R) and γc, which are essential for normal T cell development and homeostasis [
41]. TCONS_00035813, which regulated
IL-7R, was similar to other lncRNAs that affect T cell function, and its expression was gradually down-regulated with the increase in age. In conclusion, all lncRNAs related to T cell development, differentiation, and immune function in goat SMGs down-regulate the expression of target genes, which may be a major reason for the down-regulation of immune-related genes during goat submandibular gland development.
Among all lncRNA target genes that were significantly enriched in immune pathways,
MS4A1 was a gene related to B cell immune function. MS4A1 (CD20) is selectively expressed on mature B cells and most malignant B cells, and has become a clinical target for the treatment of B-cell lymphoma and some autoimmune diseases [
42]. The trans-acting lncRNA XR_001919671.1 of
MS4A1 was gradually down-regulated with the growth and development of goat SMGs, which reduced the risk of autoimmune diseases in goat SMGs to some extent. Therefore, further studies on the regulatory principle of XR_001919673-1 in the treatment of B-cell lymphoma and some autoimmune diseases may provide new insights for the treatment of B-cell lymphoma and some autoimmune diseases.
By high-throughput sequencing, we identified 17 immune-related lncRNAs by transcriptome analysis of SMGs in 1-month-old kids, 12-month-old unbred adolescent goats, and 24-month-old adult goats. The lncRNAs were all related to the immune function of T cells or B cells. The results predicted the possible changes of immune-related genes in goat SMGs with growth and development from the perspective of lncRNAs. Some of these target genes are closely related to the occurrence and development of diseases. Through further verification, these lncRNAs may become biomarkers for improving autoimmunity and disease treatment.
4.2. circRNAs Were Invoved in Immune-Ralated Pathways
With the development of RNA-sequencing and computer technology, the study of circRNAs is gradually becoming a hot topic. A variety of mammalian-derived circRNAs, including human [
43], goat [
9], mouse [
44] and pig [
45], have been identified. CircRNAs have also been identified in goat hair follicles [
46], mammary glands [
47], skeletal muscle [
9] and endometrium [
48], while studies on goat submandibular gland circRNAs have not been reported. CircRNA expression had high spatiotemporal specificity, and it is crucial to study these molecules at different development stages in different tissues. In this study, a total of 17,263 circRNAs were identified from nine libraries in three developmental stages of the goat SMGs by high-throughput sequencing. Analysis of the chromosomal origin of circRNAs revealed that all 29 autosomes of goats could produce circRNAs, with chromosome 1 producing the largest number of circRNAs, and chromosome 1 was previously found to be the main source of circRNAs in goat skeletal muscle [
9], suggesting that chromosome 1 in goats was closely related to the production of circRNAs. The length of circRNAs was mainly concentrated in the range of 100–600 nt, with the largest number between 300–400 nt. Among all circRNAs, annot_exons type circRNAs were the most numerous, and a similar phenomenon was observed in DEcircRNAs. We predicted that these types of circRNA-originating genes have a higher coding capacity and deserve our attention.
The result of DEcircRNA analyses showed that the expression pattern of submandibular gland circRNAs in adolescent and adult goats was similar, while it was quite different from that of goat kids. The enrichment analysis of DEcircRNA host genes showed that all the significantly enriched terms were biological processes, with all three of the top terms being cancer-related pathways, and all the circRNA host genes were upregulated. The phosphoinositide 3-kinase (PI3K) has the function of catalyzing the production of phosphatidylinositol-3,4,5-trisphosphate in cell survival pathways, regulating gene expression and cellular metabolism, cytoskeletal rearrangement, and binding to catalytic subunits p110α, β, δ. and γ [
49]. In this study, differentially expressed novel_circ_000481 host gene
PIK3CA (encoding p110) and novel_circ_013299 host gene
PIK3R1 (encoding p85α) were significantly enriched in the B-cell receptor signaling pathway, glioma, the FoxO signaling pathway, prostate cancer, and other pathways, which were up-regulated in adolescent goats. Engelman showed that T antigens in polyomaviruses require physical interaction with PI3K to transform cells [
50]. Overactivation of p110δ also caused T cell senescence, lymphadenopathy, and immunodeficiency [
51]. Some cancers have activating mutations in the PI3K regulatory subunit P85α (encoded by
PIK3R1). In wild-type PI3K holoenzymes, p85 inhibited p110α through intermolecular interactions, and this inhibition was relieved by a conformational change caused by the binding of the p85 amino-terminal SH2 structural domain to phosphotyrosine [
52]. Therefore, we speculate that novel_circ_000481 and novel_circ_013299 may be closely related to immune regulation in goats, and their low expression in theSMGs of goat kids may be a protection.
It’s worth noting that DEcircRNA host genes were significantly enriched in immune-related pathways. To further study the immune-related circRNAs, the top 10 DEcircRNAs for all significantly enriched immune-related pathways were screened as core immune circRNAs based on the expression level, and their target genes were
NFATC3,
LOC102189946, CR2,
TXK,
LOC102180664,
PLCG2,
ARHGAP5,
ARHGAP35,
PIGR, and
LOC100860813, respectively. Complement receptor type 2 (CR2; CD21), with an immunomodulatory effect, was found on normal T lymphocytes, which may play a role in regulating the immune response function of T cells and cellular susceptibility to lymphophilic viral infection [
53]. CD19 is a B cell-restricted membrane protein in the immunoglobulin superfamily that is associated with the antigen receptor complex. CR2 (complement receptor type 2, CD21) allows the non-immune attachment of CD19, which may facilitate the interaction of B cells with other essential cells for cell activation [
54]. Txk is an important regulator of cytokines produced by the CD4+ effector T cell population [
55]. Phospholipase Cγ2 (PLCγ2), encoded by PLCG2, is associated with human urticaria, immunodeficiency, and autoimmune diseases. Microglia-mediated innate immune responses, which were led by PLCG2 mutations, promote the development of Alzheimer’s disease [
56].
CR2,
NFATC3,
TXK, and
PLCG2 were regulated by novel_circ_011088, novel_circ_ 012271, novel_circ_004727, and novel_circ_012149, respectively. The four circRNAs were all highly expressed in goat kids and lowly expressed in adolescent and adult goats, which may indicate that the expression of immune-related genes in the SMGs of goats was down-regulated with age, and this was consistent with the analysis of DE mRNAs in goats.
P190RhoGAP-A (glucocorticoid receptor DNA-binding factor 1, or GRLF1, p190A) and p190RhoGAP-B (p190B), which were two isoforms of the p190RhoGAP protein, were encoded by
ARHGAP35 and
ARHGAP5, respectively, and p190RhoGAP was considered as a major negative regulator of RhoA. Through its role in cytoskeleton remodeling, P190RhoGAP may be related to the pathogenesis of other stress-related and neurodegenerative diseases [
57].
ARHGAP35 has also been proposed as an important new cancer gene [
58]. Similarly to P190A, the overexpression of p190B was associated with the expression of
MCT1 (multiple copies in T-cell malignancy-1, MCT1) in breast cancer [
59]. In vitro MCT1-Src-p190B interaction caused tumoral multinucleated formation in breast cancer cells [
60], which has also been demonstrated in nasopharyngeal and lung cancer. The high expression of
ARHGAP35 and
ARHGAP5 in the SMGs of adolescent goats suggested that the pathogenic genes in adolescent goats were different from those in goat kids and adult goats.
In organisms, secretory IgA (SIgA) antibodies represent the first line of antigen-specific immune defense. Most pathogens establish infection through mucous membranes, while secretory IgA (SIgA) antibodies play an “immune rejection” role in humoral defense [
61] to protect the mucosal surface from environmental pathogens and antigens, and to maintain the homeostasis of symbiotic microorganisms. The previous study results of the goat SMG transcriptome in different developmental stages showed that the expression of IgA in adolescent goats was significantly higher than that of goat kids. It was the same with novel_circ_011081, which derives from the host gene pIgR. Therefore, we predict that novel_circ_011081 may play a certain role in preventing oral infectious diseases in adolescent goats.
By transcriptome analysis, 10 immune-related circRNAs were identified from goat submandibular glands; this was the first time to analyze the immune-related circRNAs in the SMGs of goats. These data provided new insights into the function of circRNAs in the submandibular gland of goats at different developmental stages.
4.3. CeRNA Regulatory Analysis
Based on the data analyses of circRNA, lncRNA, miRNA, and mRNA of goat submandibular glands, core RNAs of each group were predicted. The expression of miR-141-x was significantly negative correlated with ITGB2, LCP2, PTPRC, SYK, and ZAP70 (r < −0.9), which indicates that the miR-141-x may play an important role in regulating the expression of core genes. The expression of PTPRC and ZAP70 was significantly and highly negatively correlated with the expression of miR-141-x, miR-29-y, and chi-miR-29b-3p.
CD45, encoded by
PTPRC, is a highly conserved protein tyrosine phosphatase receptor, which is of great significance in the development and maturation of lymphocytes, functional regulation, and signal transduction, and plays an important role in promoting the activation and development of T cells [
62]. CD45 deficiency causes T-and B-lymphocyte dysfunction, which manifests as severe combined immunodeficiency [
63,
64]. ZAP70, a member of the Syk family, is mainly involved in the initiation of T-cell receptor (TCR) signaling and subsequent T-cell activation and is a major tyrosine kinase protein [
65]. The regulation of its expression directly affected T cell activity, which further affected the activity of goat SMGs. The regulatory relationships among core RNAs in this study imply the complexity of the regulation of gene expression by non-coding RNAs.
The analyses of four RNA molecules in the goat SMGs indicated the targeting relationships of all core circRNAs. It was predicted that novel_circ_012441 could regulate the expression of ARHGAP35 in goat SMGs by sponge adsorption of chi-miR-130b-3p, chi-miR-15b-5p, and chi-miR-16b-5p. Similarly, novel_circ_013769 regulated the expression of ARHGAP5 by adsorbing chi-miR-130b-3p and miR-29-y. We predicted that regulating the expression of pathogenic genes in goat SMGs at different developmental stages might inhibit the occurrence of diseases.