- 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. - 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. - 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.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - However, to the best of our know- ledge, studies of lncRNAomics of dairy goats have been reported only in ovary, hair follicle, skeletal muscle, hypo- thalamus, and skin pigmentation tissue [10–16], and there are no studies of the lncRNAomics of dairy goat mam- mary gland tissues in different lactating periods.. - In the current study, for enhanced identification and characterization of the lncRNA profiles and function at the genome level, dairy goat mammary gland tissues in early lactation ((10th day after parturition), peak lacta- tion (90th day after parturition) and late lactation (210th day after parturition) were collected, and the distribu- tion, expression abundance and differential expression patterns of lncRNAs among different lactating periods of mammary gland development were tested and identified.. - Based on the data and bioinformatics, the action of lncRNAs on their target genes, the characteristics of mRNA transcripts, GO (gene ontology) enrichment and KEGG pathways (Kyoto Encyclopedia of Genes and Ge- nomes) were also analyzed. - From these results, we obtained a comprehensive characteristic of the lncRNA expression profile, and these results may help to elucidate the function of lncRNAs as members of the ceRNA (competing endogenous RNA) network in milk biosynthesis, transportation, and secretion or mammary gland development. - A total of and high- quality clean reads were obtained in the E, P and L librar- ies, respectively (E: early lactation, 10th day after partur- ition. - To further estimate the quality and coverage of the obtained transcripts, se- quence alignment was performed on the reference gen- ome of Capra hircus. - For the different libraries and 94.12% of the reads were mapped on the gen- ome, including unique matching reads and multiple matching reads in the E library unique matching reads and 1,602,814 multiple match- ing reads in the P library, as well as unique matching reads and 1,617,016 multiple matching reads in the L library. - Identification and genomic features of lncRNAs in the dairy goat mammary gland. - To systematically identify lncRNAs in the whole genome wide, putative lncRNAs were identified based on their evolution conservation among different species, hom- ology with known proteins, protein domains, and ORFs.. - N substrate, low quality and rRNAs (ribosomal RNA), of the 267 million clean reads, 2381 potential lncRNAs were predicted by CPC, CNCI and PFAM with their genomics position, including 1777 known lncRNAs and 604 predicted novel lncRNAs, of which 667 were in- tronic lncRNAs, 536 were intergenic lncRNAs, 326 were antisense lncRNAs, 171 were sense lncRNAs and 42 lncRNAs belonged to bidirectional lncRNAs.. - To explore the genomic features of these lncRNAs, the comparison of the characteristics was further performed with mRNAs. - The lengths of the lncRNAs ranged from 417 to 602,577 nt, and most of them (86.3%) were approximately 400~3000 nt in length. - 2d), and the median expression abundance of lncRNAs was significantly lower than those of mRNAs (Fig. - The conservation of lncRNAs was also investi- gated, which were compared with those from sheep, cows mice and humans using BLASTN version 2.2. - Of the 2381 lncRNAs in Laoshan dairy goats, 2371 lncRNAs were identified in sheep with a sequence identity greater than 80%, and in cows, humans and mice, they were 2358, 664 and 360, respectively (Fig. - Family analysis and classification as miRNA precursors of lncRNAs. - Based on evolutionary conservation, 59 known lncRNAs and 33 novel lncRNAs were classified into 53 different Table 1 Data classification in different libraries obtained by RNA sequencing. - 1 Chromosomal mapping of transcripts in the genome of Capra hircus . - a Sequence distribution of lncRNAs. - families in the Rfam database by sequence alignment.. - In all, 21 lncRNAs, including six known lncRNAs and 15 novel lncRNAs, were identified as precursors from 461 known miRNAs (Table S1).. - Differentially expressed lncRNAs and mRNAs in different lactating stages. - In total, 2381 lncRNAs were identified and expressed in at least one of the three mammary gland lactating stages. - In early lactation, compared with peak and late lactations, there were 633 differentially expressed lncRNAs, including 367 downregulated lncRNAs and 300 upregulated lncRNAs. - Between early and peak lactation, 222 lncRNAs were differentially expressed, and between early and late lactation, 512 were differentially expressed. - In peak lacta- tion, compared with early and late lactations, 684 lncRNAs were differentially expressed. - Compared with late lactation, 588 lncRNAs were differentially expressed,. - including 207 upregulated lncRNAs and 381 downregu- lated lncRNAs. - To further understand the functions of lncRNAs at the transcriptional or posttranscriptional levels, the anti- sense, up/downstream, and cis-regulated mRNAs were analyzed. - Of these RNAs, 573 lncRNAs and 1237 mRNAs were differentially expressed in mammary. - 2 Comparison of the characteristic architecture between lncRNAs and mRNAs. - f Conservation comparison of lncRNAs among different species. - Function prediction of lncRNAs and corresponding genes In the present study, we used BLAST and RNAplex to pre- dict the functions of lncRNAs and the corresponding genes.. - To further understand the biological functions of the identified lncRNAs and their genes, KEGG pathway ana- lysis was also carried out (Fig. - 3 Distribution of lncRNAs and mRNAs in different lactation periods. - a Total number of lncRNAs in different lactation periods. - b The number of differentially expressed lncRNAs between different lactation periods. - d The number of differentially expressed mRNAs between different lactation periods. - A total of 537 lncRNAs with a read number greater than 10 were screened, including up- or down- stream target genes or antimRNAs, and 1238 mRNAs were identified from differentially expressed mRNAs, which were up- or downstream of lncRNAs or their antisense chains. - Finally, based on the miRNAs, differen- tially expressed lncRNAs and related mRNAs, 1290 pairs of factors with potential regulatory relationships were se- lected and used for the ceRNA regulation network con- struction (Fig. - The results of further core model scoring analysis indicated that, two core models, miR-1247 and miR-2284r, played important regulatory functions in the ceRNA network via connecting lncRNAs and mRNAs.. - With the development of high-throughput sequencing tech- nology, an increasing number of lncRNAs have been identified, and their functions have also become increas- ingly detailed [19, 20].. - In the present study, mammary gland tissues in early, peak and late lactation periods from five dairy goats. - To better connect with our previ- ous study [17, 18], and the integrated analysis of multio- mics data in the next step, the method of mixing pools of samples in the same period was also adopted in the current study. - According to the quality control results, the construction and sequencing were successful, high quality data were obtained in three libraries, and 2381 lncRNAs and 37,249 mRNAs were identified.. - The number of lncRNAs identified in different lactation stages was also different, and some specifically expressed lncRNAs were found at a particular stage (Fig. - This finding in- dicates that lncRNAs may play a unique role in the de- velopment and lactation stages of dairy goat mammary gland tissues. - These characteristics indicate the func- tional diversity of lncRNAs, which may perform different important physiological regulatory roles in mammary gland development and lactation. - 4 GO annotation enrichment of related genes of lncRNAs. - and characterize the expression of lncRNAs in peak, late and dry lactation stages [23, 24], and lncRNAs, as an im- portant posttranscriptional regulators, participate widely in the regulation pathways related to the development and lactation of mammary glands. - Therefore, an exten- sive investigation of the functions of lncRNAs will provi- deus with a clear understanding of the lncRNA word in various biological processes.. - However, due to a lack of corresponding databases, current methods, such as the analysis of sequence charac- teristics, molecular free energy, or secondary structure, for predicting the specific functions of lncRNA genes are still imperfect, and the biological functions of the majority of lncRNAs remain vague. - From these results, it is clearly indicated that most lncRNAs together with their apcGenes and antigens participate widely in diverse biological regulation path- ways, such as HTLV-I infection, and proteoglycans in can- cer., These physiological processes are closely related to cell proliferation and apoptosis, and they are the same as those involved in the physiological cycle of mammary gland development, These genes may play a leading role. - Different colors represent different q -values, and the size of the dots indicates the number of enriched genes. - in the processes regulating mammary gland development (Figs. - In the current study, the following factors were screened to con- struct the ceRNA regulation networks: lncRNAomic and transcriptomic data, cis- and trans-mRNAs, up- and downstream mRNAs of lncRNAs, target mRNAs of miRNAs, and the bioinformatics conjoint differen- tially expressed lncRNAs and mRNAs. - 6, Table S8), and two core models, miR-1247 and miR-2248r, contained the largest number of interaction regulatory elements, indicating that they may be important regulatory factors in the lactation physiology of dairy goat mammary glands. - In summary, based on these studies, we found that lncRNAs, as core regulatory elements, play important roles in mammary gland development and lactation physiology in dairy goats. - In current study, based on the three libraries from early, peak and late lactations of dairy goat mammary gland tissues, 37,249 transcripts were obtained, including 2381 lncRNAs and 37,249 mRNAs, of which, 800 noncoding transcripts and 21,688 coding transcripts differed signifi- cantly (p ≤ 0.01) among different lactating periods. - These findings support that lncRNA, as a critical regulatory factor, widely participates in mammary gland development and lactation in dairy goats. - Our results may enlarge the foundation for the further investigation of the core mo- lecular functions of lncRNA in the physiological regula- tion of domestic animals, but may also provide some. - The mammary gland specimens in early (10th day after parturition), peak (90th day after parturition) and late (210th day after parturition) lactations, were collected surgically after general anesthesia by intramuscular injection of Xylazine Hydrochloride injection solution (Huamu Ani- mal Health Products Co., Ltd. - All mammary gland specimens were only used for scientific study, and were not released.. - Individual RNA samples collected from five dairy goat mam- mary gland tissues in the same lactating stage were thor- oughly mixed, and used further for library construction using the Illumina TruSeq Stranded RNA Kit (Illumina, USA) ac- cording to the manufacturer’s recommendations, The sam- ples were named the E library (early lactation), P library (high lactation) and L library (late lactation. - To systematically profile and identify and quantitate transcripts of dairy goat mammary gland tissues in dif- ferent lactating periods, we performed PE150 (paired- end 150) sequencing to obtain global transcripts at the whole-genome level. - Bowtie2 soft was used to scan the tran- scripts, and the rRNA, tRNA, snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), premiRNA (pre- cursor miRNA) and pseudogenes, were also recognized and removed in the next analysis. - A stringent filtering analysis pipeline was developed to identify potential lncRNAs from the assembled tran- scripts according to Wu et al transcripts that overlapped with any protein-coding exon in the sense orientation were removed. - Phylogenetic analysis of lncRNAs and mRNAs was carried out by BLASTN (E-value ≤ 1e-5, sequence iden- tity >. - Family analysis and precursor prediction of lncRNAs Infernal software [39] was used to align to the Rfam database. - Differential expression and apcGene (adjacent protein- coding genes) analysis of lncRNAs. - For differential expression, each lncRNA was identified by EdgeR version in the three libraries using an FDR no more than 0.05 and an absolute fold change no less than two as the screening conditions.. - According to the location of the lncRNAs and protein- coding genes on the Capra hircus genome (v1.01), the nearest protein-coding genes located on the 5′ upstream or 3′ downstream of a lncRNA were named apcGenes.. - All genes that were differentially expressed between dif- ferent libraries combined with apcGenes and postulate target genes were annotated and enriched in the GO database using GOseq package v1.18.0 for R [42]. - Signifi- cant pathways were enriched by KOBAS v2.0 [43] in the KEGG pathway database. - Based on the above comprehensive analysis results of the GO and KEGG of all DEGs (differential expression genes), apcGenes, and the predicted target genes of the lncRNAs, we selected genes and lncRNAs related to mammary gland development and lactation to construct the regulation network. - Family classification of lncRNAs in the Rfam database.. - Differentially expressed lncRNAs between different lactation periods.. - Predicted apcGenes of lncRNAs.. - GO annotation of related genes of lncRNAs.. - KEGG pathways of related genes of lncRNAs.. - RX, JZ and ZBJ performed the integration analysis of lncRNAs and genes. - This work was supported by the National Natural Science Foundation of China Shandong Provincial Natural Science Foundation of China (ZR2014CM029), a project of the Shandong Province Higher Educational Science and Technology Program of China (J14LF07), the Shandong Provincial Modern Agriculture Industry Technology System (SDAIT-10), and funds of the Shandong “ Double Tops ” Program (SYL2017YSTD12). - The funding body did not exert influence on the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - Global intersection of Long non-coding RNAs with processed and unprocessed Pseudogenes in the human genome. - Transcriptome analysis of the mammary gland from GH transgenic goats during involution. - Genes regulating lipid and protein metabolism are highly expressed in mammary gland of lactating dairy goats. - Screening and evaluating of long noncoding RNAs in the puberty of goats.. - Genome-wide discovery of lincRNAs with spatiotemporal expression patterns in the skin of goat during the cashmere growth cycle.. - Transcriptome sequencing to detect the potential role of long non-coding RNAs in bovine mammary gland during the dry and lactation period. - Systematic analysis of Long non-coding RNAs and mRNAs in the ovaries of Duroc pigs during different follicular stages using RNA sequencing. - Systematic identification and characterization of Long non-coding RNAs in the silkworm, Bombyx mori
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt