| Mammary glands are exocrine glands located in the breast of mammals that secrete milk to feed offspring.As the reproductive cycle occurs,mammary cells undergo repeated physiological processes of proliferation,expansion,differentiation of secretory function,and involution.Especially during the non-lactating period,mammary cells undergo involution processes such as apoptosis and autophagy,and then return to their pre-gestational state to prepare for a new round of lactation,suggesting a high degree of plasticity in mammary cells.The current research on mammary gland involution and cell remodeling in rodents and dairy cows provides the basis and reference for mammary gland development and lactation biology.However,the process of mammary gland involution and remodeling in dairy goats has not been clarified so far.Therefore,this study took the mammary gland tissues of Laoshan dairy goats at the late lactation(240 days after postpartum),the dry period(300 days after postpartum),and the late gestation(140 days after mating)as the research objects(3 samples in each period).Whole-transcriptomic sequencing of non-lactating mammary gland tissues was performed to reveal the transcriptome characteristics of non-lactating mammary gland tissue and to elucidate the molecular regulation mechanisms of involution and cell remodeling from multiple perspectives of codingRNA(mRNA)and non-codingRNA(miRNA,lncRNA,and circRNA).The research results are mainly divided into the following four aspects:1.The results of mRNA analysis showed that this study successfully constructed 9 goat mammary gland tissue transcriptome libraries,each library obtained an average of 153 million clean reads,and the alignment rate with the goat reference genome was ≥ 95%.A total of 1,381 differentially expressed genes were identified in mammary tissues of 3 different developmental stages(LL,DP and LG).Functional enrichment analysis results showed that differential genes were mainly enriched in GO terms and KEGG pathways related to cell growth,apoptosis,immunity,nutrient transport,synthesis,and metabolism.Significant enrichment of GO terms such as humoral immune response,complement activation,and neutrophil-mediated immunity(FDR < 0.05)indicated that the immune system plays an important role in maintaining the health of mammary gland tissue and clearing apoptotic cells during involution.Gene module analysis related to mammary gland development revealed that genes encoding pregnancy hormone receptor protein,cell growth factor and its receptor,and insulin-like growth factor binding protein regulate the physiological process of mammary gland involution through adaptive transcriptional changes.Further mammary gland growth and development network analysis results showed that ERBB4 was identified as the hub gene regulating mammary gland growth and development,and it was highly expressed in mammary gland tissue in late gestation.Overexpression of ERBB4 in goat mammary epithelial cells cultured in vitro can regulate the PI3K-Akt signaling pathway,reduce cell cycle G1/S phase arrest and apoptosis,and promote mammary epithelial cell proliferation.In addition,the ERBB4 also affects the expression of genes(STAT1,STAT3,STAT5 B,BCL2L11,CAPN2,and LGALS3)that initiate mammary gland involution and promote mammary cell remodeling.2.The results of miRNA analysis showed that 9 smallRNA libraries were successfully constructed in this study,and each library obtained an average of 25 million clean reads and the alignment rate with the goat reference genome was ≥ 65%.A total of 4,038 miRNA were identified,including 3,129 known miRNA and 909 newly predicted miRNA.These miRNA were annotated into 385 miRNA families,among which the mir-10,let-7,and mir-9 families had the largest number of miRNA.In addition,754 differentially expressed miRNA were identified in mammary gland tissues at 3 different stages.Cluster analysis was performed according to miRNA expression levels,and a total of 6 groups of miRNA clusters with unique expression patterns were obtained.The miRNA in cluster 1 and cluster 4 were highly expressed specifically during the dry period,and their target genes were significantly enriched in the regulation of cell morphogenesis and extracellular matrix tissue physiological processes(FDR< 0.05),indicating that the functions of these miRNA may be related to mammary gland tissue remodeling process during the dry period.The miRNA in clusters 2 and 5 were specifically low-expressed during the dry period,and their target genes were significantly enriched in the physiological processes related to the epidermal growth factor receptor signaling pathway and developmental growth regulation,and on the other hand,they were associated with cell apoptosis.Physiological processes related to immunity were also enriched(FDR < 0.05),which may be related to the extensive apoptosis of mammary epithelial cells in the mammary involution.In addition,miRNA in cluster 3 were highly expressed in LG stage,and miRNAs in cluster 6 were highly expressed in LL stage.Among them,miRNA target genes in cluster 3were enriched in physiological processes such as glandular epithelial cell differentiation,regulation of transsynaptic signaling,and columnar epithelial cell differentiation,which are associated with rapid proliferation of mammary epithelial cells and functional differentiation of lactation in late gestation.The miRNA target genes in cluster 6 were enriched in the regulation of exogenous apoptosis signaling pathways,negative regulation of macroautophagy,and cellular responses to peptide hormone stimulation,and other physiological processes.This research shows the miRNA expression characteristics of non-lactation goat mammary gland tissue and demonstrates the regulatory relationship between miRNA and their target genes related to cell growth,apoptosis,substance metabolism,etc.during mammary gland involution.3.The results of the lncRNA analysis showed that 3,074 lncRNA were identified in the mammary gland tissues of non-lactating goats.Statistical analysis of lncRNA length characteristics and exon numbers found that goat lncRNA were shorter in length,had fewer exons,and had lower expression levels compared with protein-coding genes.331 differentially expressed lncRNA were identified in the three comparison groups(LL vs.DP,DP vs.LG,and LL vs.LG),which indicated that the expression of lncRNA at the transcriptional level was changed during mammary gland involution.Interestingly,lncRNA expression was more active during the dry period compared with lactation,suggesting that lncRNA in the mammary gland are developmentally specific.GO annotation and KEGG pathway enrichment analysis showed that lncRNA can regulate the physiological processes of immune function,cell proliferation,apoptosis,hormone,substance metabolism,transport,and intercellular communication in mammary gland through multiple modes of action.Among them,cis-acting lncRNA enhanced the protection of mammary gland health during the dry period and late gestation.In addition,in the lncRNA-miRNA-mRNA network related to mammary gland development,LOC102168552 was identified as a hub lncRNA,its expression level in late gestation was higher than that in the dry period and late lactation and its expression level was significantly positively correlated with PRLR,and significantly negatively correlated with chi-mi R-324-3p(P.adj < 0.05).Overexpression of LOC102168552 in goat mammary epithelial cells cultured in vitro can activate the prolactin signaling pathway by up-regulating PRLR through competitive binding to chi-mi R-324-3p,promote cell proliferation,reduce cell cycle arrest in G1/S phase,and inhibit cell apoptosis.However,overexpression of LOC102168552 alone did not affect mammary cell growth status and prolactin signaling pathway.This suggests that LOC102168552 must rely on chi-mi R-324-3p to inhibit mammary cell apoptosis.4.The results of circRNA analysis showed that a total of 11,756 circRNA were identified in this study,of which 2,528 circRNA were expressed in all three stages.The number of exonic circRNA is the largest,and the least circRNA is antisense circRNA.circRNA source gene analysis found that 9,282 circRNA were derived from 3,889 genes,and 127 circRNA 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 diversity of circRNA source genes functions.218 differentially expressed circRNA were identified during the non-lactation period.The number of specifically expressed circRNA was the highest in DP and the least in LL.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 endogenousRNA(ceRNA)regulatory networks related to mammary development,immunity,substance metabolism,and apoptosis.These findings will help understand the regulatory role of circRNA in mammary cell involution and remodeling.In summary,this study identified a number of differentially expressed mRNA,miRNA,lncRNA,and circRNA in the mammary gland tissues of non-lactating dairy goats.It was found that genes or non-codingRNAs related to cell growth,apoptosis,immunity,nutrient transport,synthesis,and metabolism undergo adaptive transcriptional changes during the non-lactation,and they have important regulatory functions for mammary gland involution and mammary cell remodeling.In addition,this study explored the roles of ERBB4 and LOC102168552 on the growth and cell cycle of goat mammary gland cells through the PI3K-Akt signaling pathway,respectively.These findings provide insight into the molecular regulation mechanisms involved in mammary gland involution and remodeling. |
