- Transcriptome sequencing to detect the potential role of long non-coding RNAs in bovine mammary gland during the dry and lactation period. - In this study, lncRNA-seq technique was employed to compare the expression profiles of lncRNAs and mRNAs from Chinese Holstein mammary gland in dry and lactation period.. - Functional enrichment analysis on target genes of lncRNAs indicated that these genes were involved in lactation-related signaling pathways, including cell cycle, JAK-STAT, cell adhesion, and PI3K-Akt signaling pathways. - The result indicated that the lactation-associated miR-221 might interact with lncRNAs TCONS_00040268, TCONS_00137654, TCONS_00071659 and TCONS_00000352, which revealed that these lncRNAs might be important regulators for lactation cycle.. - Conclusion: This study provides a resource for lncRNA research on lactation cycle of bovine mammary gland.. - It expands our knowledge about lncRNA and miRNA biology as well as contributes to clarify the regulation of lactation cycle of bovine mammary gland.. - Keywords: lncRNA, Lactation cycle, Cow mammary gland, miR-221. - Mammary gland is an important organ for the synthesis and secretion of milk, which provides essential nutrients for human and other animal offspring. - The development and regression cycles of mammary gland include preg- nancy, lactation, and involution, which is regulated by growth factors, hormones, and coding genes [1–3]. - secretory function of mammary gland [4]. - An increasing number of studies had demonstrated that miRNAs were involved in lactation of mammary gland by regulating their target genes [14–18]. - The overexpression of miR-206 changed the expres- sion of Wnt, Tbx3 and Lef1 genes which were essential for mammary gland development, indicating that miR-206 might be a novel candidate for morphogenesis during the initiation of mammary gland formation [16]. - As a down- stream regulator of PTEN, miR-486 expressed higher during bovine high quality lactation period and could regulate the secretion of β-casein, lactose and triglyceride in BMECs, which indicated that miR-486 was required for the development of cow mammary gland [17]. - In cattle, the expression of miR-221 was found to be higher in peak lactation than in early lactation, suggesting its role in the control of endothelial cell proliferation or angiogenesis [18]. - In mice, miR-221 regulated lipid metabolism in mammary epithelial cells and was expressed differentially at various stages during mammary gland development [19]. - observation demonstrated that lncRNA Neat1 could regulate mammary gland morphogenesis and lactation by investigating the proliferation of Neat1-mutant cells in mice [26]. - However, the functions and profiles of lncRNAs in bovine mammary gland during dry and lactation period were largely unknown. - Animals and mammary gland tissue collection. - After intravenous injection of lidocaine hydrochloride, approximately 4 g of mammary gland tissues were harvested via repeated biopsies from four cows at the dry period and four cows at approximately 180 days during lactation period. - Total RNA of mammary gland tissues was isolated using Trizol reagent following the manufacturer’s instructions (Invitrogen, CA, USA). - Prediction and functional enrichment analysis of lncRNA target genes. - To reveal the potential function of lncRNAs, their target genes were predicted in trans and cis. - refers to lncRNA’s action on neighboring target genes. - www.bioinf.uni-leipzig.de/~htafer/index.html) was used to predict lncRNA target genes in trans. - The potential target genes of miR-221 were predicted by Targetscan (http://www.targetsca- n.org/vert_71. - The genes shared by the above three tools were selected as the candidate target genes.. - The cells were seeded in 24-well plates, then transfected with miR-221 mimic, mimic-NC, miR-221 inhibitor and inhibitor-NC (GenePharma, Shanghai, China) at approximately 50%. - Briefly, Lipofectamine 2000 Transfection Reagent (1 μL) was diluted in 25 μL of Opti-MEM (Invitrogen) with miR-221 mimic, mimic-NC, inhibitor, or inhibitor-NC to yield final concentrations of and 40 nM, respectively. - Overview of sequencing data in cow mammary gland A total of and raw reads were pro- duced from cow mammary glands using the Illumina Hiseq3000 platform in dry and lactation periods, respect- ively. - 0.05) were identified, such as negative regulation of cell growth (GO growth factor activity (GO:. - It was worthy to notice that the process of cell adhesion and division were closely associated with mammary gland architecture construction, maintenance, development, and lactation [41, 42].. - a The relative expression of novel transcripts and b the genome distribution of clean reads in cow mammary glands in dry and lactation periods, respectively. - Previous studies had determined that Wnt, PPAR, CAMs, PI3K-Akt, and TNF signaling pathways could regulate mammary gland development, milk fat synthesis, mas- titis, BMECs proliferation and apoptosis [43–47]. - DELs and their potential interacted miRNAs involved in lactation. - Apoptotic signaling pathway Negative regulation of growth. - GO terms revealed that significantly up-regulated genes might relate to lactation-associated processes, including calcium ion binding, negative regulation of cell growth and proliferation, gene expression, etc. - It showed that several miRNAs might interact with multiple lncRNAs, such as miR-221 interacting with lncRNAs TCONS_00032404, TCONS TCONS_00040268, TCONS_00137654, TCON S_00071659, TCONS_00143274 and TCONS_00000352.. - Coincidentally, it had demonstrated that the expression of miR-221 was found to be higher in peak lactation than in early lactation, suggesting a role in the control of endothelial cell proliferation or angiogenesis which was closely related with lactation [19], so those above mentioned lncRNAs could be considered as important candidates for lactation.. - The interaction between lncRNAs and miR-221 as well as miR-221 and ErBb3. - To verify the interaction between lncRNAs and miR-221 and the interaction between miR-221 and ErBb3, overex- pression and inhibition of miR-221 was employed in BMECs. - Our data showed that the expressions of the four lncRNAs (TCONS_00040268, TCONS_00137654, TCONS_00071659, and TCONS_00000352) decreased with the overexpression of miR-221. - miR-221 in BMECs (Fig. - 5a), which indicated that miR-221 could interact with these four lncRNAs. - Fur- thermore, the overexpression of miR-221 could reduce the expression of ErBb3 gene in miR-221 mimic group compared with mimic-NC group. - While the inhibition of miR-221 increased ErBb3 gene expression, which indicated that ErBb3 gene was the target of miR-221 (Fig. - Interaction network between the candidate lncRNAs and their potential target genes. - To reveal the potential function of these selected lncRNAs, their target genes were predicted in cis and trans roles, and we found that the mammary gland biol- ogy related genes, such as PRLR, FAS, and MAP3K7 genes, could be targeted by several lncRNAs in cis or trans (Table 3). - In addition, the network between lncRNAs and target genes was constructed. - Therefore, it could be deduced that TCONS_00075230 might involve in regulating the pro- liferation of mammary epithelial cells by interacting with IL1B gene in cow mammary gland.. - 5 The interaction between lncRNAs and miR-221 as well as miR-221 and ErBb3 gene. - (TCONS_00040268, TCONS TCONS_00071659, and TCONS_00000352) changed with the overexpression and inhibition of miR-221 b miR-221 could target ErBb3 gene by transfecting miR-221 mimic and inhibitor in BMECs. - Table 3 DELs and their potential target genes involved in lactation. - GO and KEGG analysis of lncRNAs potential target genes A total of 47 potential target genes in cis of lncRNAs (Additional file 10) were selected to carry out functional enrichment analysis. - In addition, phosphorylation regulation of mammary S6 K1 and eIF2 genes could control milk protein yield [50].. - Similarly, a total of 459 potential target genes in trans of lncRNAs (Additional file 11) were selected to perform functional enrichment analysis (Fig. - These terms were protein glycosylation (GO positive regulation of cell division (GO DNA-directed RNA polymerase III complex (GO and growth factor binding signaling pathway (GO etc. - It was worth mentioning that cell division signaling pathway had been demonstrated to be closely associated with mammary gland architecture construction and mainten- ance [42]. - Meanwhile, the KEGG results from the target genes of candidate lncRNAs in trans and cis illustrated 15 path- ways (Fig. - Combine the bioinformatics analysis of DEGs and target genes of DELs, the interaction network between proteins was produced using String software (Fig. - 6 The interaction network between candidate lncRNAs and their potential target genes. - In addition, they found most lncRNA in bovine mammary gland were downregulated compared with other tissues [58]. - 7 Functional enrichment analysis of lncRNAs target genes in cis and trans. - a Gene Ontology (GO) analysis of lncRNAs target genes in cis and trans . - The results indicated that those genes seem to play an essential role in lactation-related pathways, including phosphorylation, protein glycosylation, cytokine-mediated, and positive regulation of cell division signaling pathway, etc. - b KEGG pathway enrichment analysis of lncRNAs target genes in cis and trans . - It was well recognized that a series of genes involved in lactation initiation, maintenance as well as mammary gland growth, development and breast cancer by direct or indirect regulation . - lncRNAs and mRNAs (known as in cis) [61]. - Bio- informatics analysis of lncRNAs target genes in trans showed that these genes played an important role in some pathways, such as MAPK, PI3K-Akt, prolactin, NF-kappa B, and Toll-like receptor signaling pathways, which played important roles during mammary gland development and lactation [4, 8, 62]. - Consequently, many lncRNAs might function through targeting mRNA which played important roles in mammary gland from non-lactation to lactation period. - A series of miRNAs had been demonstrated to regu- late lactation-associated processes, including lactation cycle, milk fat accumulation, hormone receptor activity, mammary gland involution and development, and lacta- tion activity of mammary epithelial cells . - In this study, some candidate lncRNAs, including TCO NS_00040268, TCONS_00137654, TCONS_00071659, and TCONS_00000352, could interact with lactation-re- lated miR-221, and miR-221 could target ErBb3 gene. - Functional enrichment analysis of target genes and interacted miRNAs prediction of 34 lncRNAs. - Additional file 2: The expression profile of DEGs in the dry and lactation periods. - Additional file 3: The expression profile of novel lncRNAs in the dry and lactation periods. - Additional file 5: The expression profile of annotated lncRNAs in the dry and lactation periods. - Additional file 6: The expression file of novel DELs in the dry and lactation periods. - Additional file 7: The expression file of annotated DELs in the dry and lactation periods. - 9 Validation of DEGs and DELs in dry and lactation periods by qRT-PCR. - Additional file 10: Predicted target genes and mRNAs of DELs (in cis. - Additional file 11: Predicted target genes and mRNAs of DELs (in trans. - BMECs: Bovine mammary epithelial cells. - Multiple hormone interactions in the developmental biology of the mammary gland. - Stat5a is mandatory for adult mammary gland development and lactogenesis. - Signal transducer and activator of transcription 5 as a key signalling pathway in normal mammary gland developmental biology and breast cancer. - Stat5 regulates the phosphatidylinositol 3-kinase/Akt1 pathway during mammary gland development and tumorigenesis. - Transcriptomic profiles of the bovine mammary gland during lactation and the dry period. - ErbB3 drives mammary epithelial survival and differentiation during pregnancy and lactation. - Expression of miR-206 during the initiation of mammary gland development. - MicroRNA expression patterns in the bovine mammary gland are affected by stage of lactation.. - miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development. - Regulation of mammary epithelial cell homeostasis by lncRNAs. - A noncoding RNA is a potential marker of cell fate during mammary gland development. - The long noncoding RNA Neat1 is required for mammary gland development and lactation. - Mammary gland growth factors: roles in normal development and in cancer. - Cell adhesion in mammary gland biology and neoplasia - preface. - J Mammary Gland Biol. - The importance of cell division in udder development and lactation. - Diverse regulation of mammary epithelial growth and branching morphogenesis through noncanonical Wnt signaling. - Keeping abreast with long non-coding RNAs in mammary gland development and breast cancer. - MiR-103 controls milk fat accumulation in goat (Capra hircus) mammary gland during lactation
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