Transcriptome profiling of the nonlactating mammary glands of dairy goats reveals the molecular genetic mechanism of mammary cell remodeling.

The mammary gland redevelops to the prepregnancy state during involution, which shows the mammary cells have the characteristics of remodeling. The rapidity and degree of mammary gland involution vary across species (e.g., between model organism mice and dairy livestock). However, the molecular genetic mechanism of involution and remodeling of goat mammary gland has not yet been clarified. This work investigated the structural changes and transcriptome characteristics of the mammary gland tissue of nonlactating dairy goats during the late lactation (LL), the dry period (DP), and late gestation (LG). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed significant changes in the structure of the nonlactating goat mammary gland, and obvious cell apoptosis occurred at LL and DP. Sequencing identified 1,381 genes that are differentially expressed in mammary gland tissue at the 3 developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism exhibited adaptive transcriptional changes to meet the needs of a new set of mammary gland lactation functions. The significant enrichment of Gene Ontology terms such as humoral immune response, complement activation, and neutrophil-mediated immunity indicates that the innate immune system plays an important role in maintaining the health of degenerative mammary glands and eliminating apoptotic cells. The peroxisome proliferator-activated receptor signaling pathway plays an important regulatory role in lipid metabolism, especially the adaptive changes in expression of genes encoded lipid transport and enzymes, which promote the formation of milk fat during the lactation. The mammary gland development gene module revealed that pregnancy hormone receptors, cell growth factors and their receptors, and genes encoding insulin-like growth factor binding proteins regulate the physiological process of mammary gland involution through adaptive transcriptional changes. Interestingly, ERBB4 was identified as the hub gene of the network that regulates mammary gland growth and development. Overexpression of ERBB4 in mammary epithelial cells cultured in vitro can reduce cell cycle arrest in G1/S phase and apoptosis by regulating the PI3K/Akt signaling pathway and promote the proliferation of mammary epithelial cells. The gene ERBB4 also affects the expression of genes that initiate mammary gland involution and promote mammary gland remodeling. These findings contribute to an in-depth understanding of the molecular mechanisms involved in mammary gland involution and remodeling.

Transcriptome Analysis of Goat Mammary Gland Tissue Reveals the Adaptive Strategies and Molecular Mechanisms of Lactation and Involution.

To understand how genes precisely regulate lactation physiological activity and the molecular genetic mechanisms underlying mammary gland involution, this study investigated the transcriptome characteristics of goat mammary gland tissues at the late gestation (LG), early lactation (EL), peak lactation (PL), late lactation (LL), dry period (DP), and involution (IN) stages. A total of 13,083 differentially expressed transcripts were identified by mutual comparison of mammary gland tissues at six developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism make adaptive transcriptional changes to meet the needs of mammary lactation. Notably, platelet derived growth factor receptor beta (PDGFRB) was screened as a hub gene of the mammary gland developmental network, which is highly expressed during the DP and IN. Overexpression of PDGFRB in vitro could slow down the G1/S phase arrest of goat mammary epithelial cell cycle and promote cell proliferation by regulating the PI3K/Akt signaling pathway. In addition, PDGFRB overexpression can also affect the expression of genes related to apoptosis, matrix metalloproteinase family, and vascular development, which is beneficial to the remodeling of mammary gland tissue during involution. These findings provide new insights into the molecular mechanisms involved in lactation and mammary gland involution.

Genome­wide integrated analysis demonstrates widespread functions of lncRNAs in mammary gland development and lactation in dairy goats.

BACKGROUND: The mammary gland is a unique organ for milk synthesis, secretion and storage, and it undergoes cyclical processes of development, differentiation, lactation and degeneration. At different developmental periods, the biological processes governing mammary gland physiology and internal environmental homeostasis depend on a complex network of genes and regulatory factors. Emerging evidence indicates that lncRNAs have arbitrarily critical functions in regulating gene expression in many organisms; however, the systematic characteristics, expression, and regulatory roles of lncRNAs in the mammary gland tissues of dairy goats have not been determined. RESULT: In the present study, we profiled long noncoding RNA (lncRNA) expression in the mammary gland tissues of Laoshan dairy goats (Capra hircus) from different lactation periods at the whole-genome level, to identify, characterize and explore the regulatory functions of lncRNAs. A total of 37,249 transcripts were obtained, of which 2381 lncRNAs and 37,249 mRNAs were identified, 22,488 transcripts, including 800 noncoding transcripts and 21,688 coding transcripts, differed significantly (p ≤ 0.01) among the different lactation stages. The results of lncRNA-RNA interaction analysis showed that six known lncRNAs belonging to four families were identified as the precursors of 67 known microRNAs; 1478 and 573 mRNAs were predicted as hypothetical cis-regulation elements and antisense mRNAs, respectively. GO annotation and KEGG analysis indicated that the coexpressed mRNAs were largely enriched in biological processes related to such activities as metabolism, immune activation, and stress,., and most genes were involved in pathways related to such phenomena as inflammation, cancer, signal transduction, and metabolism. CONCLUSIONS: Our results clearly indicated that lncRNAs involved in responses to stimuli, multiorganism processes, development, reproductive processes and growth, are closely related to mammary gland development and lactation.

Genome-wide analysis reveals signatures of selection for important traits in domestic sheep from different ecoregions.

BACKGROUND: Throughout a long period of adaptation and selection, sheep have thrived in a diverse range of ecological environments. Mongolian sheep is the common ancestor of the Chinese short fat-tailed sheep. Migration to different ecoregions leads to changes in selection pressures and results in microevolution. Mongolian sheep and its subspecies differ in a number of important traits, especially reproductive traits. Genome-wide intraspecific variation is required to dissect the genetic basis of these traits. RESULTS: This research resequenced 3 short fat-tailed sheep breeds with a 43.2-fold coverage of the sheep genome. We report more than 17 million single nucleotide polymorphisms and 2.9 million indels and identify 143 genomic regions with reduced pooled heterozygosity or increased genetic distance to each other breed that represent likely targets for selection during the migration. These regions harbor genes related to developmental processes, cellular processes, multicellular organismal processes, biological regulation, metabolic processes, reproduction, localization, growth and various components of the stress responses. Furthermore, we examined the haplotype diversity of 3 genomic regions involved in reproduction and found significant differences in TSHR and PRL gene regions among 8 sheep breeds. CONCLUSIONS: Our results provide useful genomic information for identifying genes or causal mutations associated with important economic traits in sheep and for understanding the genetic basis of adaptation to different ecological environments.

Three slow skeletal muscle troponin genes in small-tailed Han sheep (Ovis aries): molecular cloning, characterization and expression analysis.

To explore the basic characteristics and expressing profile of the three slow skeletal muscle troponin genes TNNC1 (Troponin C type 1), TNNI1 (troponin I type 1) and TNNT1 (troponin T type 1). Three purebred Dorper sheep and another three purebred small-tailed Han sheep were selected. The sequence of the genes from the small-tailed Han sheep was cloned using rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction; The characteristics of the predicted amino acids sequences were analyzed using bioinformatics analysis software; Gene expression analyses were performed using quantitative reverse transcription PCR. The full-length cDNA sequences of the genes were 707, 898, and 1001 bp, respectively, and were submitted to GenBank under accession numbers KR153938, KT218688 and KT218690. The three predicted proteins were predicted to be hydrophilic, non-secretory proteins and contain several phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted proteins were relatively conserved in mammals. The expression results of the three genes in eight tissues of Dorper and small-tailed Han sheep revealed that the three genes had a similar mRNA expression pattern, whereas distinct differences were observed among the eight tissues of the two sheep species. We cloned the full-length cDNA of the three genes, analyzed the amino acid sequences, and determined the expression levels of the genes. These results might play important roles in facilitating the future research of the three genes.

De novo assembly and characterization of the skeletal muscle transcriptome of sheep using Illumina paired-end sequencing.

OBJECTIVE: In order to enrich the ovine genome and provide a basis for future molecular genetics and functional genomics analyses in sheep, we used de novo assembly to establish transcriptomes of skeletal muscle tissues of Dorper and Small-tailed Han sheep. RESULTS: A total of 103,058,824 clean Illumina paired-end sequencing reads from the two libraries were assembled into 145,524 unigenes in a de novo project. There were 5718 unigenes showing differential expression between the two transcriptomes, and 7437 coding SSRs were exploited. After further assembly, we identified a total of 70,348 all-unigenes with an average length of 863 bp; 35,201 of these all-unigenes could be annotated in the Nr database, and 12,219 were found in the clusters of orthologous groups database. Gene ontology searches indicated cell and binding as the main terms. Among 258 Kyoto Encyclopedia of Genes and Genomes database pathways, protein and amino acid metabolism pathways were the most commonly identified. CONCLUSION: We analyzed the ovine muscle transcriptome using high-throughput sequencing technology. Many unigenes were assembled and numerous molecular markers and differential expressed unigenes were identified.

Identification and Function Prediction of Novel MicroRNAs in Laoshan Dairy Goats.

MicroRNAs are a class of endogenous small RNAs that play important roles in post-transcriptional gene regulation by directing degradation of mRNAs or facilitating repression of target gene translation. In this study, three small RNA cDNA libraries from the mammary gland tissues of Laoshan dairy goats (Capra hircus) were constructed and sequenced, individually. Through Solexa high-throughput sequencing and bioinformatics analysis, we obtained 50 presumptive novel miRNAs candidates, and 55,448 putative target genes were predicted. GO annotations and KEGG pathway analyses showed the majority of target genes were involved in various biological processes and metabolic pathways. Our results discovered more information about the regulation network between miRNAs and mRNAs and paved a foundation for the molecular genetics of mammary gland development in goats.

[Association of polymorphisms of exon 2 of GOLA-DQA2 gene with blood immune traits in goats].

The polymorphisms of exon 2 of GOLA-DQA2 gene were analyzed by PCR-RFLP technique in Laiwu black goats, Lubo goats, and Boer goats; and the effects of genotypes of GOLA-DQA2 gene on blood immune traits were estimated. The results showed that four genotypes were detected in the three goats. Polymorphic sites were detected at base positions 77, 79, 80, and 169 in exon 2 of GOLA-DQA2 gene. The effects of breed were major effect. In Lubo goats, red blood count (RBC) with genotype AB was significantly higher than that with genotype BB (P<0.05), and white blood count (WBC) with genotype AB was significantly higher than that with genotype BC (P<0.05). Hematocrit (HCT) in Lubo goats with genotype AB was significantly higher than that with genotypes BB and BC (P<0.05). WBC-large cell ratio (W-LCR) in Lubo goats with genotype BC was significantly higher than that with genotype BB (P<0.05). In Laiwu black goats and Boer goats, there was certain degree of association between genotypes and blood immune traits, but it was not significant. Genotype AB and BC was an important factor affecting RBC, WBC, W-LCR, and so on. It was concluded from these results that GOLA-DQA2 was the gene affecting the blood immune traits, which provides a basis for disease resistance breeding of goats.

Identification and Characterization of circRNAs in Non-Lactating Dairy Goat Mammary Glands Reveal Their Regulatory Role in Mammary Cell Involution and Remodeling.

This study conducted transcriptome sequencing of goat-mammary-gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages to reveal the expression characteristics and molecular functions of circRNAs during mammary involution. A total of 11,756 circRNAs were identified in this study, of which 2528 circRNAs were expressed in all three stages. The number of exonic circRNAs was the largest, and the least identified circRNAs were antisense circRNAs. circRNA source gene analysis found that 9282 circRNAs were derived from 3889 genes, and 127 circRNAs' source genes were unknown. Gene Ontology (GO) terms, such as histone modification, regulation of GTPase activity, and establishment or maintenance of cell polarity, were significantly enriched (FDR < 0.05), which indicates the functional diversity of circRNAs' source genes. A total of 218 differentially expressed circRNAs were identified during the non-lactation period. The number of specifically expressed circRNAs was the highest in the DP and the lowest in LL stages. These indicated temporal specificity of circRNA expression in mammary gland tissues at different developmental stages. In addition, this study also constructed circRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) regulatory networks related to mammary development, immunity, substance metabolism, and apoptosis. These findings help understand the regulatory role of circRNAs in mammary cell involution and remodeling.

Identification and characterization of microRNA in the dairy goat (Capra hircus) mammary gland by Solexa deep-sequencing technology.

microRNAs (miRNAs) perform critical roles in various biological and metabolic processes by regulating gene expression at the post-transcriptional level. To investigate the functional roles of miRNAs in the lactating mammary gland of Capra hircus, a library was constructed from the lactating mammary glands of Laoshan dairy goats (C. hircus) during early lactation. The miRNA expression profiles were systematically screened, and miRNAs were identified and characterized using Solexa deep-sequencing technology and bioinformatics. As a result, a total of 18,031,615 clean reads were obtained representing 305,711 unique sRNAs. A total of 12,086,616 sRNAs representing 3,701 unique sRNAs matched the known Bos taurus miRNA precursors in miRBase 17.0, and 300 known miRNAs and 15 miRNA were discovered. In addition, 131 novel miRNAs sequences were also obtained, and 147,703 putative targets were predicted. GO and KEGG pathway analysis showed that the majority of targets were involved in cellular processes and metabolic pathways. The 290 known miRNAs, 14 miRNA and 38 novel miRNAs were validated by sequencing a second library that was constructed from the same tissues as the first library. Our study provided the first large-scale identification and characterization of miRNAs in the mammary gland tissue of the dairy goat. The results indicate that the regulation of miRNA-mediated gene expression occurs during early lactation in dairy goats. This study significantly enriches the C. hircus miRNA repertoire and provides a reference for the elucidation of complex miRNA-mediated regulatory networks for gene expression in the physiology and developmental progression of the lactating mammary gland.

MAPK1 Is Regulated by LOC102188416/miR-143-3p Axis in Dairy Goat Mammary Epithelial Cells.

MicroRNA-143-3p (miR-143-3p) is one of the miRNAs involved in the growth of goat mammary epithelial cells (GMECs). In this study, Illumina/Solexa sequencing was performed to establish the lncRNA database in Laoshan dairy goats. Using the lncRNA database, long noncoding RNAs (lncRNAs) regulated by miR-143-3p were screened. In total, 4899 lncRNAs were identified, with 173 lncRNAs being differentially expressed in all three replicates. The target genes of the differentially expressed lncRNAs were annotated in GO terms and KEGG pathways. Among the differentially expressed lncRNAs, lncRNA LOC102188416 was predicted to sponge miR-143-3p and share MAPK1 as a common target gene with miR-143-3p, which was validated by dual luciferase reporter assay system and qRT-PCR. The miR-143-3p mimic significantly lowered the relative luciferase activity of psiCHECK2-LOC102188416 wildtype vector but not mutated vector, suggesting that lncRNA LOC102188416 might be a sponge of miR-143-3p, which was verified by the promotion role of lncRNA LOC102188416 siRNA (siR-LOC102188416) in the expression of miR-143-3p. It was shown that the expression of MAPK1 was downregulated by either miR-143-3p mimic or siR-LOC102188416, indicating that miR-143-3p and lncRNA LOC102188416 had a coregulatory effect on MAPK1 expression. The co-transfection of miR-143-3p inhibitor with siR-LOC102188416 reversed the decrease of MAPK1 expression regulated by siR-LOC102188416 alone, strengthening the existence of lncRNA LOC102188416/miR-143-3p/MAPK1 axis in GMECs of Laoshan dairy goats.

Identification and functional analysis of m6A in the mammary gland tissues of dairy goats at the early and peak lactation stages.

N6-methyladenosine (m6A) is the most common reversible epigenetic RNA modification in the mRNA of all higher eukaryotic organisms and plays an important role in the regulation of gene expression and cell function. In this study, m6A-modified methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptome sequencing (RNA-seq) were used to identify the key genes with m6A modification during mammary gland development and lactation in dairy goats. The results showed that m6A methylation occurred at 3,927 loci, which were significantly enriched in the 3' untranslated region (3'UTR) and the termination codon region. In the early stage and peak stage of lactation, m6A methylation occurred extensively in mammary tissues, and a total of 725 differentially expressed m6A-modified genes were obtained, all negatively correlated with mRNA expression. In addition, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that different methylated genes were mainly involved in the growth and apoptosis of mammary epithelial cells through signaling pathways, such as the mitogen-activated protein kinase (MAPK) and phospholipase D pathways, and then affected the development and lactation of mammary gland. All in all, we identified and analyzed the methylation events related to the development and lactation regulation of mammary gland at the early and peak lactation stages, and provided a theoretical basis to reveal the physiological regulatory system of mammary gland development and lactation in dairy goats.

Characterization of long noncoding RNA in nonlactating goat mammary glands reveals their regulatory role in mammary cell involution and remodeling.

Long noncoding RNA (lncRNA) can regulate mammary gland development and lactation physiological activities. However, the molecular genetic mechanisms of lncRNA in mammary gland involution and cell remodeling remain unclear. This work analyzed the expression characteristics and molecular functions of lncRNA in goat mammary gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages. Sequencing results showed that 3074 lncRNAs were identified in non-lactating goat mammary gland tissue. Statistical analysis of lncRNA length characteristics and exon number found that goat lncRNAs were shorter in length, had fewer exons, and significantly lower expression levels than those of protein-coding genes. 331 differentially expressed lncRNAs were identified in the three comparison groups (LLvsDP, DPvsLG, and LLvsLG), which indicated that the lncRNAs expression at the transcriptional level were changed during mammary involution. Interestingly, lncRNAs were more actively expressed during the dry period compared to lactation, suggesting that lncRNAs in mammary glands are developmentally specific. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that lncRNAs could regulate immune function, cell proliferation, apoptosis, hormones, substance metabolism, transport, and intercellular communication in the mammary gland through various action modes. Among them, cis-acting lncRNAs enhanced the protection of mammary gland health during the dry period and late gestation. The above reflects the particular mechanisms of lncRNA to adapt to the developmental needs of mammary involution and remodeling. Furthermore, in the lncRNA-miRNA-mRNA network associated with mammary gland development, the expression of LOC102168552 was higher in late gestation than in the dry period and late lactation. Its expression was positively correlated with PRLR and negatively correlated with chi-miR-324-3p. Overexpression of LOC102168552 in goat mammary epithelial cells cultured in vitro could up-regulate PRLR to activate the prolactin signaling pathway by competitively binding to chi-miR-324-3p, promoting cell proliferation, reducing cell cycle arrest in the G1 / S phase, and inhibiting apoptosis. However, overexpression of LOC102168552 alone did not affect mammary cell growth status and the prolactin signaling pathway. This indicates that LOC102168552 must rely on chi-miR-324-3p to inhibit mammary cell apoptosis. In conclusion, the above analysis revealed that lncRNAs in goat mammary tissue are differentially expressed at different stages of involution. As expected, lncRNAs adaptively regulate various physiological activities during mammary gland involution through multiple modes of action, in preparation for a new round of lactation. These findings provide a reference and help further understand the regulatory role of lncRNAs in mammary cell involution and remodeling.

High-Throughput Sequencing Reveals Transcriptome Signature of Early Liver Development in Goat Kids.

As a vital metabolic and immune organ in animals, the liver plays an important role in protein synthesis, detoxification, metabolism, and immune defense. The primary research purpose of this study was to reveal the effect of breast-feeding, weaning transition, and weaning on the gene expression profile in the goat kid liver and to elucidate the transcriptome-level signatures associated with liver metabolic adaptation. Therefore, transcriptome sequencing was performed on liver tissues, which was collected at 1 day (D1), 2 weeks (W2), 4 weeks (W4), 8 weeks (W8), and 12 weeks (W12) after birth in Laiwu black goats at five different time-points, with five goats at each time point. From 25 libraries, a total of 37497 mRNAs were found to be expressed in goat kid livers, and 1271 genes were differentially expressed between at least two of the five time points. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that these genes were annotated as an extracellular region fraction, exhibiting monooxygenase activity, positive regulation of T cell activation, mitotic spindle mid-region assembly, cytokinesis, cytoskeleton-dependent cytokinesis, regulation of cytokinesis, regulation of lymphocyte proliferation, and so on. In addition, it mainly deals with metabolism, endocrine, cell proliferation and apoptosis, and immune processes. Finally, a gene regulatory network was constructed, and a total of 14 key genes were screened, which were mainly enriched for cell growth and development, endocrine, immune, and signal transduction-related pathways. Our results provide new information on the molecular mechanisms and pathways involved in liver development, metabolism, and immunity of goats.

Differentially Expressed MiRNAs of Goat Submandibular Glands Among Three Developmental Stages Are Involved in Immune Functions.

Submandibular glands (SMGs) are one of the primary components of salivary glands in goats. The proteins and biologically active substances secreted by the SMGs change with growth and development. Our previous studies showed that most of the differentially expressed genes in the SMGs of goats at different developmental stages are involved in immune-related signaling pathways, but the miRNA expression patterns in the same tissues are unknown. The aim of this study was to reveal the expression profile of miRNAs at three different developmental stages, detect differentially expressed miRNAs (DE miRNAs) and predict disease-related DE miRNAs. SMG tissue samples were collected from groups of 1-month-old kids, 12-month-old maiden goats and 24-month-old adult goats (three samples from each group), and high-throughout transcriptome sequencing was conducted. A total of 178, 241 and 7 DE miRNAs were discovered between 1-month-old kids and 12-month-old maiden goats, between 1-month-old kids and 24-month-old adult goats, and between 12-month-old maiden goats and 24-month-old adult goats, respectively. Among these DE miRNAs, 88 DE miRNAs with medium or high expression levels (TPM ≥50) were classified into five expression pattern clusters. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that some of the predicted target genes of the DE miRNAs in the five clusters were enriched in disease-related GO terms and pathways. MiRNA target genes in significant pathways were significantly enriched in Hepatitis B (FDR = 9.03E-10) and Pathways in cancer (FDR = 4.2E-10). Further analysis was performed with a PPI network, and 10 miRNAs were predicted to play an important role in the occurrence and prevention of diseases during the growth and development of goats.

Characterization of the microRNA Expression Profiles in the Goat Kid Liver.

The liver is the largest digestive gland in goats with an important role in early metabolic function development. MicroRNAs (miRNA) are crucial for regulating the development and metabolism in the goat liver. In the study, we sequenced the miRNAs in the liver tissues of the goat kid to further research their regulation roles in early liver development. The liver tissues were procured at 5-time points from the Laiwu black goats of 1 day (D1), 2 weeks (W2), 4 weeks (W4), 8 weeks (W8), and 12 weeks (W12) after birth, respectively with five goats per time point, for a total of 25 goats. Our study identified 214 differential expression miRNAs, and the expression patterns of 15 randomly selected miRNAs were examined among all five age groups. The Gene ontology annotation results showed that differential expression miRNA (DE miRNA) target genes were significantly enriched in the fatty acid synthase activity, toxin metabolic process, cell surface, and antibiotic metabolic process. The KEGG analysis result was significantly enriched in steroid hormone synthesis and retinol metabolism pathways. Further miRNA-mRNA regulation network analysis reveals 9 differently expressed miRNA with important regulation roles. Overall, the DE miRNAs were mainly involved in liver development, lipid metabolism, toxin related metabolism-related biological process, and pathways. Our results provide new information about the molecular mechanisms and pathways in the goat kid liver development.

Transcriptome analysis reveals potential immune function-related regulatory genes/pathways of female Lubo goat submandibular glands at different developmental stages.

BACKGROUND: The submandibular glands, as major salivary glands, participate in rumen digestion in goats. Sialic acid, lysozyme, immunoglobulin A (IgA), lactoferrin and other biologically active substances secreted in the submandibular glands were reported in succession, which suggests that the submandibular gland may have immune functions in addition to participating in digestion. The aim of this study was to map the expression profile of differentially expressed genes (DEGs) at three different stages by transcriptome sequencing, screen immune-related genes and pathways by bioinformatics methods, and predict the immune function of submandibular glands at different developmental stages. METHODS: Nine submandibular gland tissue samples were collected from groups of 1-month-old kids, 12-month-old adolescent goats and 24-month-old adult goats (3 samples from each group), and high-throughput transcriptome sequencing was conducted on these samples. The DEGs among the three stages were screened and analysed. Key genes and signalling pathways were selected via protein-protein interaction (PPI) network analysis. RESULTS: The results revealed 2,706, 2,525 and 52 DEGs between 1-month-old and 12-month-old goats, between 1-month-old and 24-month-old goats, and between 12-month-old and 24-month-old goats, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that most of the DEGs were enriched in immune- related GO terms and pathways. Based on functional enrichment analysis and network analysis, 10 genes (PTPRC, CD28, SELL, LCP2, MYC, LCK, ZAP70, ITGB2, SYK and CCR7), two signalling pathways (the T cell receptor signalling pathway and the NF-κß signalling pathway) and eight GO terms (T cell receptor signalling pathway, neutrophil mediated immunity, B cell mediated immunity, regulation of alpha-beta T cell activation, positive regulation of T cell proliferation, regulation of leukocyte differentiation, positive regulation of antigen receptor-mediated signalling pathway, positive regulation of lymphocyte proliferation) that may play key roles in the immune functions of the goat submandibular glands at different developmental stages were identified. Moreover, we found that eight antibacterial peptide-encoding genes were downregulated in the tuberculosis and salivary secretion pathways, while all immunoglobulins were upregulated in 10 immune system pathways. These findings indicate that the submandibular glands may be important immunological organs during the growth process of goats and that the immune function of these glands gradually weakens with age up to 12 months but remains relatively stable after 12 months of age. Overall, this study will improve our understanding of transcriptional regulation related to goat submandibular gland immune function.

Characterization of microRNA profiles in the mammary gland tissue of dairy goats at the late lactation, dry period and late gestation stages.

MicroRNAs (miRNAs) play an important role in regulating mammary gland development and lactation. We previously analyzed miRNA expression profiles in Laoshan dairy goat mammary glands at the early (20 d postpartum), peak (90 d postpartum) and late lactation (210 d postpartum) stages. To further enrich and clarify the miRNA expression profiles during the lactation physiological cycle, we sequenced miRNAs in the mammary gland tissues of Laoshan dairy goats at three newly selected stages: the late lactation (240 d postpartum), dry period (300 d postpartum) and late gestation (140 d after mating) stages. We obtained 4038 miRNAs and 385 important miRNA families, including mir-10, let-7 and mir-9. We also identified 754 differentially expressed miRNAs in the mammary gland tissue at the 3 different stages and 6 groups of miRNA clusters that had unique expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that GO terms such as mammary gland development (GO:0030879) and mammary gland morphogenesis (GO:0060443) and important signaling pathways, including the insulin signaling pathway (chx04910), hippo signaling pathway (chx04390) and estrogen signaling pathway (chx04915), were enriched. We screened miRNAs and potential target genes that may be involved in the regulation of lactation, mammary gland growth and differentiation, cell apoptosis, and substance transport and synthesis and detected the expression patterns of important genes at the three stages. These miRNAs and critical target genes may be important factors for mammary gland development and lactation regulation and potentially valuable molecular markers, which may provide a theoretical reference for further investigation of mammary gland physiology.

chi-miR-143-3p Promotes Apoptosis of Mammary Gland Epithelial Cells from Dairy Goats by Targeting Ndfip1.

The mammary gland is an important organ for lactation in dairy goats. Mammary gland development and lactation functions are primarily regulated by natural hormones and certain crucial regulatory factors. Nedd4 family-interacting protein 1 (Ndfip1) can specifically bind to neural precursor cell-expressed, developmentally downregulated protein 4 (Nedd4) family members to participate in ubiquitination, which in turn regulates a range of biological processes in the body. However, the effects of Ndfip1 expression regulation at the post-transcriptional level on the development of mammary gland cells have not been previously reported. To study the regulation of Ndfip1 at post-transcriptional level, the overexpression and interference vectors of Ndfip1 were constructed, and co-transfected into the primary mammary gland epithelial cells cultured in vitro with miR-143 mimics and inhibitor. Dual luciferase reporter gene system, real-time quantitative polymerase chain reaction, western blotting, cholecystokinin octapeptide assays, and flow cytometry were used to identify their regulation and function. As a result, Ndfip1 was targeted and regulated by miR-143, which influences the development of mammary gland epithelial cells in dairy goats cultured in vitro. This study will lay an experimental foundation for further understanding the functions of Ndfip1 and miR-143.

Transcriptome Analysis of Dairy Goat Mammary Gland Tissues from Different Lactation Stages.

The mammary gland is a unique organ involved in lactation in dairy livestock, and its development and lactation ability are affected by both genetic and environmental factors. To explore the molecular regulatory mechanisms of these factors, this study used high-throughput sequencing technology and bioinformatics methods to systematically analyze the transcriptome of Laoshan dairy goat mammary gland tissues from different lactation stages. From three libraries, 36,336,892, 36,469,596, and 35,759,380, pure sequences were obtained, with 25,292, 23,665 and 27,220 expressed genes, respectively, resulting in a total of 14,892 nonredundant differentially expressed genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these genes were annotated to biological regulation, cellular processes, metabolic processes, cells, organelles, binding, catalytic activity and transcriptional activity, and so on. The genes were mainly involved in cellular processes, environmental information processes, genetic information processes, human diseases, metabolism, and organ systems. Finally, 20 genes related to mammary gland development and lactation were screened to construct a gene regulatory network. These findings support the involvement of the constructed network in the regulation of mammary gland development and lactation, and they not only lay the foundation to further investigate and screen the main genes or molecular genetic markers controlling the development of the mammary gland and lactation ability of dairy goats but also increase the in-depth understanding of the lactation physiology of dairy goats.