Project description:The sex-linked dwarf (SLD) chicken is an ideal model system for understanding growth hormone (GH)-action and growth hormone receptor (GHR) function because of its recessive mutation in the GHR gene. Skeletal muscle mass is reduced in the SLD chicken with a smaller muscle fiber diameter. Our previous study has presented the mRNA and miRNA expression profiles of the SLD chicken and normal chicken between embryo day 14 and seven weeks of age. However, the molecular mechanism of GHR-deficient induced muscle mass loss is still unclear, and the key molecules and pathways underlying the GHR-deficient induced muscle mass loss also remain to be illustrated. Here, by functional network analysis of the differentially expressed miRNAs and mRNAs between the SLD and normal chickens, we revealed that let-7b, miR-128 and the MAPK pathway might play key roles in the GHR-deficient induced muscle mass loss, and that the reduced cell division and growth are potential cellular processes during the SLD chicken skeletal muscle development. Additionally, we also found some genes and miRNAs involved in chicken skeletal muscle development, through the MAPK, PI3K-Akt, Wnt and Insulin signaling pathways. This study provides new insights into the molecular mechanism underlying muscle mass loss in the SLD chickens, and some regulatory networks that are crucial for chicken skeletal muscle development.
Project description:Skeletal muscle fibers are primarily categorized into oxidative and glycolytic fibers, and the ratios of different myofiber types are important factors in determining livestock meat quality. However, the molecular mechanism for determining muscle fiber types in chickens was hardly understood. In this study, we used RNA sequencing to systematically compare mRNA and microRNA transcriptomes of the oxidative muscle sartorius (SART) and glycolytic muscle pectoralis major (PMM) of Chinese Qingyuan partridge chickens. Among the 44,705 identified mRNAs in the two types of muscles, 3,457 exhibited significantly different expression patterns, including 2,364 up-regulated and 1,093 down-regulated mRNAs in the SART. A total of 698 chicken miRNAs were identified, including 189 novel miRNAs, among which 67 differentially expressed miRNAs containing 42 up-regulated and 25 down-regulated miRNAs in the SART were identified. Furthermore, function enrichment showed that the differentially expressed mRNAs and miRNAs were involved in energy metabolism, muscle contraction, and calcium, peroxisome proliferator-activated receptor (PPAR), insulin and adipocytokine signaling. Using miRNA-mRNA integrated analysis, we identified several candidate miRNA-gene pairs that might affect muscle fiber performance, viz, gga-miR-499-5p/SOX6 and gga-miR-196-5p/CALM1, which were supported by target validation using the dual-luciferase reporter system. This study revealed a mass of candidate genes and miRNAs involved in muscle fiber type determination, which might help understand the molecular mechanism underlying meat quality traits in chickens.
Project description:A deletion mutation in the growth hormone receptor (GHR) gene results in the inhibition of skeletal muscle growth and fat deposition in dwarf chickens. In this study, microarray techniques were used to detect the miRNA and mRNA expression profiles of 14-day-old embryo and 7-week-old chicken skeletal muscle of deletion-type dwarf chickens and normal-type chickens. Skeletal muscle tissues of Dwarf recessive White Rock chickens and normal recessive White Rock chickens were used to make the microarray assay. Results show the expression of miR-1623 and miR-181b in 14-day-old embryos and of let-7b and miR-128 in 7-week-old chickens. let-7b was the only miRNA found to be completely complementary to its target in the 3'UTR of GHR and inhibited GHR gene expression. KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway analysis and RT-PCR verified that there were three main signalling pathways regulating the skeletal muscle growth and fat deposition of chickens influenced by the let-7b-regulated GHR gene. The suppression of the cytokine signalling 3 (SOCS3) gene was found to be involved in the signalling pathway of adipocytokines. We found that let-7b is the critical miRNA involved in the regulation of the GHR gene. SOCS3 plays a critical role in the network regulating skeletal muscle growth and fat deposition via let-7b-mediated GHR gene expression. Two groups were analyzed in the array assay: one group consisted of normal recessive White Rock 7-week-old chicken leg muscle tissues, and the other group consisted of dwarf recessive White Rock 7-week-old chicken leg muscle tissues. The control samples were labeled as A1b, A2b, A3b, and the dwarf chicken samples were labeled as B1b, B2b, and B3b. 9 total chickens per breed, 3 chickens used per breed for each sample. 523 mature miRNA sequences were assembled and integrated into the LC miRNA microarray design, and different expression miRNAs were measured on the 7000HT Fast Real-Time PCR system. REPLACE This submission represents the miRNA profiling component of the study.
Project description:BACKGROUND: Functional studies have demonstrated that microRNAs (miRNAs or miRs) play critical roles in a wide spectrum of biological processes including development and disease pathogenesis. To investigate the functional roles that miRNAs play during chicken skeletal muscle development, the miRNA transcriptomes of skeletal muscles from broiler and layer chickens were profiled using Solexa deep sequencing. RESULTS: Some miRNAs have multiple isoforms and several miRNAs* are present at higher levels than their corresponding miRNAs. Thirty three novel and 189 known chicken miRNAs were identified using computational approaches. Subsequent miRNA transcriptome comparisons and real-time PCR validation experiments revealed 17 miRNAs that were differentially expressed between broilers and layers, and a number of targets of these miRNAs have been implicated in myogenesis regulation. Using integrative miRNA target-prediction and network-analysis approaches an interaction network of differentially expressed and muscle-related miRNAs and their putative targets was constructed, and miRNAs that could contribute to the divergent muscle growth of broiler and layer chickens by targeting the ACVR2B gene were identified, which can causes dramatic increases in muscle mass. CONCLUSIONS: The present study provides the first transcriptome profiling-based evaluation of miRNA function during skeletal muscle development in chicken. Systematic predictions aided the identification of potential miRNAs and their targets, which could contribute to divergent muscle growth in broiler and layer chickens. Furthermore, these predictions generated information that can be utilized in further research investigating the involvement of interaction networks, containing miRNAs and their targets, in the regulation of muscle development.
Project description:Pig is an important agricultural animal for meat production and provides a valuable model for many human diseases. Functional studies have demonstrated that microRNAs (miRNAs) play critical roles in almost all aspects of skeletal muscle development and disease pathogenesis. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for porcine microRNAome (miRNAome) during 10 skeletal muscle developmental stages including 35, 49, 63, 77, 91 dpc (days post coitum) and 2, 28, 90, 120, 180 dpn (days postnatal) using Solexa sequencing technology. Our results extend the repertoire of pig miRNAome to 247 known miRNAs processed from 210 pre-miRNAs and 297 candidate novel miRNAs through comparison with known miRNAs in the miRBase. Expression analysis of the 15 most abundant miRNAs in every library indicated that functional miRNAome may be smaller and tend to be highly expressed. A series of muscle-related miRNAs summarized in our study present different patterns between myofibers formation phase and muscle maturation phase, providing valuable reference for investigation of functional miRNAs during skeletal muscle development. Analysis of temporal profiles of miRNA expression identifies 18 novel candidate myogenic miRNAs in pig, which might provide new insight into regulation mechanism mediated by miRNAs underlying muscle development.
Project description:The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3'UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3'UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens.
Project description:The different skeletal muscle fiber types exhibit distinctively different physiological and metabolic properties, and have been linked to both human metabolic diseases and meat quality traits in livestock. Circular RNAs (circRNAs) are a new class of endogenous RNA regulating gene expression, but regulatory mechanisms of skeletal muscle fibers involved in circRNAs remain poorly understood. Here, we constructed circRNA expression profiles of three fast-twitch biceps femoris (Bf) and three slow-twitch soleus (Sol) muscles in pigs using RNA-seq and identified 16,342 distinct circRNA candidates. Notably, 242 differentially expressed (DE) circRNAs between Bf and Sol muscles were identified, including 105 upregulated and 137 downregulated circRNAs, and are thus potential candidates for the regulation of skeletal muscle fiber conversion. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of host genes of DE circRNAs revealed that host genes were mainly involved in skeletal muscle fiber-related GO terms (e.g., muscle contraction, contractile fiber part, and Z disk) and skeletal muscle fiber-related signaling pathways (e.g., AMPK and cGMP-PKG). We also constructed co-expression networks of DE circRNA-miRNA-mRNA using previously acquired high-throughput sequencing mRNA and miRNA data, from which 112 circRNA-miRNA and 95 miRNA-mRNA interactions were identified. Multiple circRNAs essentially serve as a sponge for miR-499-5p, which is preferentially expressed in slow-twitch muscle and reduces the severity of Duchenne muscular dystrophy (DMD). Taken together, a series of novel candidate circRNAs involved in the growth and development of porcine skeletal muscle was identified. Furthermore, they provide a comprehensive circRNA resource for further in-depth research on the regulatory mechanisms of circRNA in the formation of skeletal muscle fiber, and may provide insights into human skeletal muscle diseases.
Project description:Pig is one of the major dietary protein sources for human consumption, from which muscle is the largest protein origin. However, molecular mechanisms concerning early porcine embryonic muscle development distinctions between pig breeds are still unclear. In this study, an integrated analysis of transcriptome and miRNAome was conducted using longissimus dorsi muscle of 4 early embryonic stages around the primary myofiber formation time (18-, 21-, 28-, and 35-d post coitus) from 2 pig breeds (Landrace [LR] and Wuzhishan [WZS]) differing in meat mass. The global miRNA/mRNA expression profile showed that WZS prepared for myogenic developmental processes earlier than LR. After identifying and analyzing the interaction network of top 100 up-/down-regulated miRNA and their target genes, we were able to find 3 gene clusters: chromatin modification-related (Chd2, H3f3a, Chd6, and Mll1), myogenesis-related (Pax3, Pbx1, Mef2a, and Znf423), and myosin component-related (Mylk, Myo5a, Mylk4, Myh9, and Mylk2) gene clusters. These genes may involve in miRNA-gene myogenic regulatory network that plays vital role in regulating distinct early porcine embryonic myogenic processes between LR and WZS. In summary, our study reveals an epigenetic-mediated myogenic regulatory axial that will help us to decipher molecular mechanisms concerning early porcine embryonic muscle development distinctions between pig breeds.
Project description:Intramuscular fat deposition or marbling is essential for high quality beef. The molecular mechanism of adipogenesis in skeletal muscle remains largely unknown. In this study, we isolated Platelet-derived growth factor receptor ? (PDGFR?) positive progenitor cells from fetal bovine skeletal muscle and induced into adipocytes. Using miRNAome sequencing, we revealed that bta-miR-23a was an adipogenic miRNA mediating bovine adipogenesis in skeletal muscle. The expression of bta-miR-23a was down-regulated during differentiation of PDGFR?+ progenitor cells. Forced expression of bta-miR-23a mimics reduced lipid accumulation and inhibited the key adipogenic transcription factor peroxisome proliferative activated receptor gamma (PPAR?) and CCAAT/enhancer binding protein alpha (C/EBP?). Whereas down-regulation of bta-miR-23a by its inhibitors increased lipid accumulation and expression of C/EBP?, PPAR? and fatty acid-binding protein 4 (FABP4). Target prediction analysis revealed that ZNF423 was a potential target of bta-miR-23a. Dual-luciferase reporter assay revealed that bta-miR-23a directly targeted the 3'-UTR of ZNF423. Together, our data showed that bta-miR-23a orchestrates early intramuscular adipogeneic commitment as an anti-adipogenic regulator which acts by targeting ZNF423.
Project description:There is a lack of understanding surrounding the molecular mechanisms involved in the development of chicken skeletal muscle in the late postnatal stage, especially in the regulation of breast muscle development related genes, pathways, miRNAs and other factors. In this study, 12 cDNA libraries and 4 small RNA libraries were constructed from Gushi chicken breast muscle samples from 6, 14, 22, and 30 weeks. A total of 15,508 known transcripts, 25,718 novel transcripts, 388 known miRNAs and 31 novel miRNAs were identified by RNA-seq in breast muscle at the four developmental stages. Through correlation analysis of miRNA and mRNA expression profiles, it was found that 417, 370, 240, 1,418, 496, and 363 negatively correlated miRNA-mRNA pairs of W14 vs. W6, W22 vs. W6, W22 vs. W14, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparisons, respectively. Based on the annotation analysis of these miRNA-mRNA pairs, we constructed the miRNA-mRNA interaction network related to biological processes, such as muscle cell differentiation, striated muscle tissue development and skeletal muscle cell differentiation. The interaction networks for signaling pathways related to five KEGG pathways (the focal adhesion, ECM-receptor interaction, FoxO signaling, cell cycle, and p53 signaling pathways) and PPI networks were also constructed. We found that ANKRD1, EYA2, JSC, AGT, MYBPC3, MYH11, ACTC1, FHL2, RCAN1, FOS, EGR1, and FOXO3, PTEN, AKT1, GADD45, PLK1, CCNB2, CCNB3 and other genes were the key core nodes of these networks, most of which are targets of miRNAs. The FoxO signaling pathway was in the center of the five pathway-related networks. In the PPI network, there was a clear interaction among PLK1 and CDK1, CCNB2, CDK1, and GADD45B, and CDC45, ORC1 and MCM3 genes. These results increase the understanding for the molecular mechanisms of chicken breast muscle development, and also provide a basis for studying the interactions between genes and miRNAs, as well as the functions of the pathways involved in postnatal developmental regulation of chicken breast muscle.