Project description:A series of complex processes regulate muscle development, and lncRNAs play essential roles in the regulation of skeletal myogenesis. Using RNA sequencing, we profiled the lncRNA expression during goat (Capra hircus) skeletal muscle development, which included seven stages across fetal 45 (F45), 65 (F65), 90 (F90), 120 (F120), 135 (F135) days, born for 24 h (B1) and 90 (B90) days. A total of 15,079 lncRNAs were identified in the seven stages, and they were less conservative with other species (human, cow, and mouse). Among them, 547 were differentially expressed, and they divided the seven stages into three functional transition periods. Following weighted gene co-expression network analysis (WGCNA), five lncRNA modules specific for developmental stages were defined as three types: 'Early modules', 'late modules', and 'individual-stage-specific modules'. The enrichment content showed that 'early modules' were related to muscle structure formation, 'late modules' participated in the 'p53 signaling pathway' and other pathways, the F90-highly related module was involved in the 'MAPK signaling pathway', and other pathways. Furthermore, we identified hub-lncRNA in three types of modules, and LNC_011371, LNC_ 007561, and LNC_001728 may play important roles in goat skeletal muscle. These data will facilitate further exploration of skeletal muscle lncRNA functions at different developmental stages in goats.
Project description:MicroRNAs (miRNAs) are indispensable for the regulation of skeletal muscle. We performed RNA sequencing (RNA-seq) to establish a comprehensive miRNA profiling of goats in seven stages, namely, 45- (F45), 65- (F65), 90- (F90), 120- (F120), and 135-day (F135) fetuses, newborn (B1), and 90-day-old (B90) kids. In total, 421 known miRNAs and 228 goat novel miRNAs were identified in the data, and the average abundance of 19 miRNAs in seven stages exceeds 10,000 reads per million. Furthermore, 420 differentially expressed miRNAs (DEmiRNAs) were identified in all comparison group at seven stages, 80 of which were uniquely differentially expressed in the B1 and B90 comparison groups. Pathway analysis indicated that this group was associated with the release of muscle hypertrophy and regulation of myoblast proliferation. Besides, 305 DEmiRNAs were clustered into three significantly enriched profiles (profiles 11, 16, and 19). Function analysis revealed that profile 16 was related to muscle hypertrophy and differentiation. Profile 11 was involved in multiple enzyme activities and metabolic processes in muscle cells. And profile 19 was involved in material transport and structural stability. Two highly expressed miRNAs and three key miRNAs (chi-miR-328-3p, chi-miR-767, and chi-miR-150) of these profiles were verified to be consistent with the data by quantitative real-time PCR. These results provided a catalog of goat muscle-associated miRNAs, allowing us to better understand the transformation of miRNA roles during mammalian muscle development.
Project description:BACKGROUND:Circular RNA (circRNA) is produced during the splicing of mRNA (in addition to linear splicing) and is part of the gene regulatory network. The temporal expression patterns the different developmental stages were inseparable from these molecules' function. RESULTS:Skeletal muscles of Anhui white goat (AWG) across seven fetal to postnatal development stages were sequenced and 21 RNA sequencing libraries were constructed. We thereby identified 9090 circRNAs and analyzed their molecular properties, temporal expression patterns, and potential functions at the different stages. CircRNAs showed complexities and diversity of formation as the same host gene produces multiple isoforms of these nucleic acids with different expression profiles. The differential expression of 2881 circRNAs (DECs, P?<?0.05) was identified and four were randomly selected and validated by qPCR. Moreover, 1118 DECs under strict selected (SDECs, |log2FC|?>?2 and P-adj value?<?0.01) showed 4 expression trends (Clusters 0, 19, 16 and 18). Cluster 0 molecules had increasing expression at all stages with effects on muscle through metabolism, regulation of enzyme activity, and biosynthesis. Cluster 16 circRNAs had high expression in the early and late stages and are involved in "Wnt signaling pathway", "AMPK signaling pathway" and others. Cluster 18 molecules were mainly expressed at F120 and participate in "cytoskeletal protein binding", "Notch signaling pathway" and so on. Cluster 19 circRNAs were down-regulated at all stages and related to muscle structure and development. Lastly, the SDECs divided the period of skeletal muscle development into three transitional stages: stage 1 (F45 to F90), which related to muscle satellite cell proliferation and muscle fiber structure; stage 2 (F90 to B1), in which the attachment of the cytoplasmic surface to the actin cytoskeleton initiates; and stage 3, which involved the "cGMP-PKG signaling pathway". Moreover, the paraffin sections messages also validated that there are three transitional stages of skeletal muscle development. CONCLUSION:Our current study provides a catalog of goat muscle-related circRNAs that can stratify skeletal muscle development fetus 45?days to newborn 90?days into three developmental stages. These findings better our understanding of functional transitions during mammalian muscle development.
Project description:Long noncoding RNAs (lncRNAs) have emerged as essential regulators of skeletal myogenesis, but few myogenesis-associated lncRNAs have been identified and our understanding of their regulatory mechanisms remains limited, particularly in goat. Here, we identified a novel lncRNA, TCONS_00006810 (named lncR-125b), from our previous lncRNA sequencing data on fetal (45, 60, and 105 days of gestation, three biological replicates for each point) and postnatal (3 days after birth, n = 3) goat skeletal muscle, and found that it is highly expressed in skeletal muscle and gradually upregulated during skeletal muscle satellite cell (SMSC) differentiation in goat. Notably, overexpression of lncR-125b accelerated the expression of myogenic differentiation 1 (MyoD 1) and myogenin (MyoG), and the formation of myotubes, and knockdown of lncR-125b showed opposite effects in SMSCs. Results of dual-luciferase assay and quantitative real-time polymerase chain reaction revealed that lncR-125b acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2), a critical regulator of skeletal myogenesis, is a direct target gene of miR-125b. Further analyses showed that lncR-125b negatively regulates miR-125b expression and positively regulates IGF2 expression in SMSCs. Mechanistically, lncR-125b promotes SMSC differentiation by functioning as a competing endogenous RNA (ceRNA) for miR-125b to control IGF2 expression. These findings identify lncR-125b as a novel noncoding regulator of muscle cell differentiation and skeletal muscle development in goat.
Project description:Skeletal muscle development is a complex biological process regulated by numerous genes and non-coding RNAs, such as microRNAs (miRNAs). In the current study, we made use of the deep sequencing data from Jianzhou Da'er goat longissimus dorsi sampled on days 45, 60, and 105 of gestation, as well as day three after birth to identify miRNAs that regulate goat skeletal myogenesis, and examine their temporal expression profiles. A total of 410 known goat miRNAs, 752 miRNA homologs and 88 novel miRNAs were identified across four stages. Besides three myomiRs, the abundance of 17 miRNAs, including chi-miR-424, chi-miR-542-3p and chi-miR-136-5p was more than 10,000 reads per million mapped reads (RPM), on average. Furthermore, 50 miRNAs with more than 100 RPM clustered at the imprinted DLK1-DIO3 locus on chromosome 21 and showed similar expression patterns, indicating that these miRNAs played important roles in skeletal myogenesis of goats. Based on pairwise comparisons, 221 differentially expressed (DE), known miRNAs were identified across four stages. GO and KEGG analyses of the genes targeted by the DE miRNAs revealed the significantly enriched processes and pathways to be consistent with temporal changes of skeletal muscle development across all sampled stages. However, follow-up experimental studies were required to explore functions of these miRNAs and targets underlying skeletal myogenesis.
Project description:Alternative splicing (AS) is a fundamental regulatory process in all higher eukaryotes. However, AS landscapes for a number of animals, including goats, have not been explored to date. Here, we sequenced 60 samples representing 5 tissues from 4 developmental stages in triplicate using RNA-seq to elucidate the goat AS landscape. In total, 14,521 genes underwent AS (AS genes), accounting for 85.53% of intron-containing genes (16,697). Among these AS genes, 6,342 were differentially expressed in different tissues. Of the AS events identified, retained introns were most prevalent (37.04% of total AS events). Functional enrichment analysis of differential and specific AS genes indicated goat AS mainly involved in organ function and development. Particularly, AS genes identified in leg muscle were associated with the "regulation of skeletal muscle tissue development" GO term. Given genes were associated with this term, four of which (NRG4, IP6K3, AMPD1, and DYSF) might play crucial roles in skeletal muscle development. Further investigation indicated these five genes, harbored 13 ASs, spliced exclusively in leg muscle, likely played a role in goat leg muscle development. These results provide novel insights into goat AS landscapes and a valuable resource for investigation of goat transcriptome complexity and gene regulation.
Project description:Intramuscular fat (IMF) content and fatty acid composition of longissimus dorsi muscle (LM) change with growth, which partially determines the flavor and nutritional value of goat (Capra hircus) meat. However, unlike cattle, little information is available on the transcriptome-wide changes during different postnatal stages in small ruminants, especially goats. In this study, the sequencing reads of goat LM tissues collected from kid, youth, and adult period were mapped to the goat genome. Results showed that out of total 24 689 Unigenes, 20 435 Unigenes were annotated. Based on expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), 111 annotated differentially expressed genes (DEGs) were identified among different postnatal stages, which were subsequently assigned to 16 possible expression patterns by series-cluster analysis. Functional classification by Gene Ontology (GO) analysis was used for selecting the genes showing highest expression related to lipid metabolism. Finally, we identified the node genes for lipid metabolism regulation using co-expression analysis. In conclusion, these data may uncover candidate genes having functional roles in regulation of goat muscle development and lipid metabolism during the various growth stages in goats.
Project description:MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding ribonucleic acids (RNAs) that regulate gene expression by binding to their complementary sequences. Recent years, a great deal of miRNAs which highly-enriched in skeletal muscle have been identified, which can influence multiple facets of muscle development and function through their regulation of key genes controlling myogenesis. However, to date no miRNAs have been reported to modulate muscle development in goat. Total RNAs from the xuhuai goats longissimus thoracis at fetal and six month old stages were used to construct small RNA libraries for Solexa SBS technology sequencing. In the small RNA profile, a total of 15,627,457 clean reads were obtained from the fetal goat library and 15,593,721 clean reads from the six month old goat library. There are 471 conserved miRNAs overlapped in both libraries, of which 343 miRNAs were differential expressed. We identified 122 novel miRNAs in the fetal caprine library and 53 novel miRNAs in the six month old-caprine library. Overall design: Examination of Chinese Xuhuai goat miRNAs by deep sequencing
Project description:Goat is an important agricultural animal for meat production. Functional studies have demonstrated that microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on miRNAs expression profiles have been performed in various animals, relatively limited information about goat muscle miRNAs has been reported. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for expression profiles of caprine miRNAs during two developmental stages of skeletal muscles: fetal stage and six month-old stage. As a result, 15,627,457 and 15,593,721 clean reads were obtained from the fetal goat library (FC) and the six month old goat library (SMC), respectively. 464 known miRNAs and 83 novel miRNA candidates were identified. Furthermore, by comparing the miRNA profile, 336 differentially expressed miRNAs were identified and then the potential targets of the differentially expressed miRNAs were predicted. To understand the regulatory network of miRNAs during muscle development, the mRNA expression profiles for the two development stages were characterized and 7322 differentially expressed genes (DEGs) were identified. Then the potential targets of miRNAs were compared to the DEGs, the intersection of the two gene sets were screened out and called differentially expressed targets (DE-targets), which were involved in 231 pathways. Ten of the 231 pathways that have smallest P-value were shown as network figures. Based on the analysis of pathways and networks, we found that miR-424-5p and miR-29a might have important regulatory effect on muscle development, which needed to be further studied. This study provided the first global view of the miRNAs in caprine muscle tissues. Our results help elucidation of complex regulatory networks between miRNAs and mRNAs and for the study of muscle development.
Project description:This study found that miR-27 is expressed in muscle and regulates muscle proliferation and differentiation. We explored the function and regulatory mechanism of miR-27b in goat muscle proliferation and differentiation. Compared with the Boer goat, higher expression of miR-27b was observed in all of the collected muscle tissues of Anhuai goat, excluding the kidney, whereas the opposite expression pattern was observed for Pax3, which showed lower expression in Anhuai goat. Expression of miR-27b decreased gradually during the proliferation of skeletal muscle satellite cells in Anhuai goat and increased during differentiation; however, the expression pattern of Pax3 was opposite. The regulatory activity of miR-27b demonstrated that miR-27b inhibited the proliferation of skeletal muscle satellite cells, but promoted their differentiation. Moreover, function research demonstrated that Pax3 negatively regulated myogenic differentiation of goat skeletal muscle satellite cells, but accelerated their proliferation. The results of a dual-luciferase reporter analysis showed that miR-27b directly targeted the 3'-untranslated regions of Pax3 mRNA, and western blot and immunofluorescence staining analyses showed that miR-27b inhibited expression of the Pax3 protein. In goats, miR-27b can regulate myogenic proliferation and differentiation by targeting Pax3.