INTRODUCTION
Although litter size of sow has made a significant improvement during last two decades, the problem of growth performance and mortality in piglets has become prominent. During gestation and lactation, the growth performance and health status of piglets have been critical factors impacting reproductive performance of modern sows [
1]. Sow milk performance is a major limiting factor and contributes to suboptimal growth and survival of piglets [
2]. The quality and quantity of milk in sows are highly variable, for example, the colostrum yield is reported to range from <1 kg to 8.5 kg [
3,
4]. To enhance the sow lactation ability, it is necessary to understand the mammary gland development, and screen their regulatory factors [
5]. In 2016, Balzani et al [
6] reported the heritability of udder morphology in crossbreed sows (Large White×Meishan), namely,
h2: 0.1 to 0.56. Generally, from colostrogenesis to lactogenesis, mammary glands might undergo significant functional differentiation for swine parturition and colostrum occurrence processes. In 2018, Palombo et al [
7] used the RNA sequencing (RNA-seq) of mammary gland to detect the candidate genes impacting swine parturition and lactation in crossbred sows (Danish Landrace×Yorkshire).
Long non-coding RNA (lncRNA) is a recently identified class of non-protein coding transcripts in eukaryotes with a minimum length of 200 nt [
8], and it plays an important regulatory role in biological processes [
9]. Many lncRNAs have been reported along with the depth and quality of RNA-seq [
10]. In swine, many potential regulatory lncRNAs have been identified from various tissues, such as intramuscular adipose [
11,
12], longissimus dorsi muscle [
13], preadipocytes [
14], and porcine endometrium [
15]. In 2018, Liang et al [
9] built a systematic
Sus scrofa lncRNA database named lncRNAnet that contained 53,468
S. scrofa lncRNAs with their sequence characteristics, genomic locations, conservation, overlapping single nucleotide polymorphism (SNP) and quantitative trait loci, and transcript abundance across nine tissues (fat, heart, kidney, liver, lung, muscle, ovarium, spleen, and testis). However, the identification of lncRNAs regulating milk traits from sow mammary gland is still lacking.
In present study, we identified lncRNAs of sow mammary gland at different stages from 14 days prior to parturition to day 1 after parturition according to the published RNA-seq data, and detected the differentially expressed (DE) lncRNAs. Further, validation of genome wide association study (GWAS) signals, functional annotation and weighted gene co-expression network analysis (WGCNA) were conducted to predict the regulatory functions of DE lncRNAs. Our results will pave the way for a better understanding of lncRNA functions in swine parturition and colostrum occurrence processes.
DISCUSSION
In this study, we systematically analyzed the RNA-seq data of 15 swine mammary gland samples collected at days 14, 10, 6, and 2 before (−) parturition to day 1 after (+) parturition and identified a total of 286 DE lncRNAs targeting 256 genes. Further, we found that these DE lncRNAs had significant association with sow lactation, and were involved in delivery and lactation development, milk lipid metabolism, and immune function of colostrum, and 18 lncRNAs targeting 20 genes which were proposed to be the candidates involved in lactation of sow.
At parturition, the preparation for copious milk synthesis and secretion have begun [
7,
26], and the mammary gland can reach the greatest degree of structural development. In this study, we performed the validation of GWAS signals, and showed that the DE lncRNAs were significantly enriched with GWAS signals of sow milk ability, which indicated that these dynamically changed lncRNAs in different periods of mammary gland progression may be involved in milk ability. We examined the expression pattern of DE lncRNAs, most of the lncRNAs expressions were strongly changed from day −6 to day +1. Further, 18 promising functional lncRNAs targeting 20 genes were mainly identified in −2 vs −14 and 1 vs −14 groups, which reflected a strong activation of many metabolic processes before and after parturition. In the WGCNA, 20 promising functional DE target genes were involved in the significant modules, which were highly associated with the mammary gland samples at day −14, day −2, and day +1. Hence, our results were consistent with the concept: before and after farrowing with the formation of colostrum, mammary glands immediately undergo strong functional differentiation [
5].
In general, lncRNAs exert regulatory functions at different levels of gene expression to influence the tumor growth, cell-cycle, and apoptosis [
24,
27]. For example, lncRNA H19 is involved in regulation of high glucose-induced apoptosis through targeting VDAC1 [
28]. Here, 11 lncRNA-mRNA target pairs,
XLOC_020627-ACTN4,
ENSSSCG00000051193-ADCY1,
XLOC_025150-CSN3,
ENSSSCG00000042618-SMO,
XLOC_963181-PTK7,
ENSSSCG00000051701-MPDZ,
XLOC_018030-NPR1,
XLOC_025146-CSN2,
ENSSSCG00 000041015-ATP2C2,
ENSSSCG00000046607-PRKAG2, and
ENSSSCG00000049698-ACTB, were involved in delivery and lactation metabolism, such as tight junctions, oxytocin, development of the mammary gland and lactation. Tight junctions of mammary gland from the pregnant animal are leaky, undergoing closure around parturition to become the impermeable tight junctions of the lactating animal [
29]. In dairy cattle, after parturition, the start of copious milk production requires the closure of tight junctions to form the blood-milk barrier and prevent paracellular transfer of blood constituents into milk (such as lactate dehydrogenase and serum albumin) and vice versa (such as appearance of α-lactalbumin in blood) [
30]. Oxytocin is a nonapeptide hormone that has a central role in the regulation of parturition and lactation [
31], and its best-known and most well-established roles are stimulation of uterine contractions during parturition and milk release during lactation [
32]. Development of the mammary gland occurs in defined stages, which are connected to sexual development and reproduction (embryonic, prepubertal, pubertal, pregnancy, lactation, and involution) [
33]. Lactation is a highly demanding lipid synthesis and transport process that is crucial for the development of newborn mammals [
34]. Embryo development ending in birth or egg hatching term was defined as the process whose specific outcome is the progression of an embryo over time, from zygote formation until the end of the embryonic life stage, and for mammals it is usually considered to be birth. The above reports suggested that these GO and KEGG enrichments were related with the parturition and lactation. Hence, we proposed that these lncRNAs in 11 lncRNA-mRNA target pairs might be regulated their target genes to impact the delivery and lactation processes.
Additionally, we found eight lncRNA-mRNA target pairs, XLOC_020627-ACTN4, ENSSSCG00000051193-ADCY1, ENSSSCG00000042618-SMO, XLOC_963181-PTK7, XLOC_ 026156-CD36, XLOC_018030-NPR1, XLOC_010589-ACSL3, and ENSSSCG00000046607-PRKAG2, were enriched in peroxisome proliferator-activated receptors (PPARs), regulation of lipolysis in adipocytes, cellular response to lipid, and adipocytokine signaling pathways, that might be related with the milk components metabolism.
For the piglets, colostrum plays an important role in ensuring their survival, growth and health by providing energy, nutrients, immunoglobulins, growth factors and many other bioactive components and cells [
35]. In the present study, we found that 12 lncRNA-mRNA target pairs,
XLOC_020627-ACTN4,
ENSSSCG00000042618-SMO,
XLOC_963181-PTK7,
ENSSSCG00000011196-GALNT15,
ENSSSCG00000041987-C6H1orf210,
ENSSSCG00000041987-CDC20,
ENSSSCG 00000041015-ATP2C2,
ENSSSCG00000046607-GALNTL5,
ENSSSCG00000042000-SNRPD1,
XLOC_002435-FIBCD1,
ENSSSCG00000049698-ACTB, and
ENSSSCG000000482 64-GALNT7, were involved in leukocyte transendothelial migration, response to retinoic acid, drug binding, mucin type O-glycan biosynthesis, regulation of defense response to virus, and positive regulation of ubiquitin-protein transferase activity enrichments, which might impact the immune function of colostrum.
Based on these advantages, we proposed these 18 lncRNAs (XLOC_020627, ENSSSCG00000051193, XLOC_025150, ENSSSCG00000042618, XLOC_963181, ENSSSCG000000 51701, XLOC_018030, XLOC_025146, ENSSSCG00000041015, ENSSSCG00000046607, ENSSSCG00000049698, XLOC_ 026156, XLOC_010589, ENSSSCG00000011196, ENSSSCG 00000041987, ENSSSCG00000042000, XLOC_002435, and ENSSSCG00000048264) as the promising candidates for swine parturition and lactation. Our network results of 18 lncRNAs, their 20 target genes and corresponding pathways further clearly showed the functions of the candidate lcnRNAs and their target genes. There is a high probability that these lncRNAs and genes interact closely and thus act as key regulators in mammary gland, thus, future work should focus on the potential roles of them in sow lactation.
Sow milk production is the major factor which can limit growth and survival of piglets. To enhance milk performance, it is necessary to understand the process of mammary gland morphogenesis and to identify the regulatory factors of mammary development. In this study, we identified 1,084 lncRNAs in swine mammary gland using RNA-seq data, and 286 were DE lncRNAs in five lactation stages. Further, the enrichment analysis of GWAS signals for sow milk ability trait validated that the identified DE lncRNAs had significant association with milk ability trait. Integrated analysis of the DE lncRNAs expression pattern examination, targets prediction, function annotation and WGCNA, we proposed that 18 lncRNAs (such as XLOC_020627, ENSSSCG00000051193, XLOC_025150, ENSSSCG00000042618, XLOC_963181, ENSSSCG000000 51701, XLOC_018030, and XLOC_025146) targeting 20 genes (such as ACTN4, ADCY1, CSN3, SMO, CSN2, PRKAG2, FIBCD1, and GALNT7) as promising candidates which were involved in swine parturition and colostrum occurrence processes. These results provided a new insight for exploring critically regulatory factors involved in reproductive performance of sow.