Role In Myogenesis Research Articles

438 DNA methylation patterns and transcriptional regulation during pig fetal skeletal muscle development

Abstract Fetal development is controlled by a complex cascade of highly regulated and coordinated gene expression patterns. Epigenetic mechanisms have important roles in regulating development and differentiation. Among such mechanisms, DNA methylation exhibits context-specific associations with gene expression and has been shown to be highly dynamic during developmental processes. We performed whole-genome bisulfite sequencing (WGBS) to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-d gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45,739 differentially methylated regions (DMRs) between stages, and the majority (n = 34,232) were hypomethylated at 70 vs. 41 dg. Developmental DMRs exhibited feature-specific enrichment in gene regulatory regions, as well as in regions proximal to micro-RNAs (miRNAs) that have known roles in myogenesis. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and transcript abundance. We surveyed myogenic regulatory factor (MRF) genes to determine if differential methylation was present in expected genomic regions. Within the MYF5 and MYF6 locus, MYF5 was significantly promoter-hypermethylated at 70 dg, whereas MYF6 was significantly hypomethylated upstream of its transcription start site. MYF5 is the earliest MRF to be expressed and primarily functions in myoblast proliferation and determination, while MYF6 functions in muscle cell differentiation. Thus, these patterns were consistent with expected downregulation of MYF5 and upregulation of MYF6 as muscle development progresses and demonstrate that differential methylation is evident at myogenic transcription factors. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We also show that developmental DMRs are enriched for GWAS SNPs associated with muscle physiology and meat quality traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics in pig fetal skeletal muscle and reveal putative cis-regulatory elements governed by epigenetic processes during porcine myogenesis.

IGFBP7 promotes the proliferation and differentiation of primary myoblasts and intramuscular preadipocytes in chicken

Though it is well known that insulin-like growth factor (IGF) binding protein 7 (IGFBP7) plays an important role in myogenesis and adipogenesis in mammals, its impact on the proliferation, differentiation, and lipid deposition in chicken primary myoblasts (CPM) and intramuscular preadipocytes remains unexplored. In the present study, we firstly examined the correlation between SNPs within the genomic sequence of the IGFBP7 gene and carcass and blood chemical traits in a F2 resource population by genetic association analysis, and found that a significant correlation between the SNP (4_49499525) located in the intron region of IGFBP7 and serum high-density lipoproteins (HDL). We then examined the expression patterns of IGFBP7 across different stages of proliferation and differentiation in CPMs and intramuscular preadipocytes via qPCR, and explored the biological functions of IGFBP7 through gain- and loss-of-function experiments and a range of techniques including qPCR, CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and Oil Red O staining to detect the proliferation, differentiation, and lipid deposition in CPMs and intramuscular preadipocytes. We ascertained that the expression levels of the IGFBP7 gene increased as cell differentiation progresses in CPMs and intramuscular preadipocytes, and that IGFBP7 promotes the proliferation and differentiation of these cells, as well as facilitates intracellular lipid deposition. Furthermore, we investigated the regulatory mechanism of IGFBP7 expression by using co-transfection strategy and dual-luciferase reporter assay, and discovered that the myogenic transcription factors (MRF), myoblast determination factor (MyoD) and myogenin (MyoG), along with the adipocyte-specific transcription factor (TF) CCAAT/enhancer-binding protein α (C/EBPα), can bind to the core transcription activation region of the IGFBP7 promoter located 500 bp upstream from the transcription start site, thereby promoting IGFBP7 transcription and expression. Taken together, our study underscores the role of IGFBP7 as a positive regulator for myogenesis and adipogenesis, while also elucidating the functional and transcriptional regulatory mechanisms of IGFBP7 in chicken skeletal muscle development and intramuscular adipogenesis.

Reprint of: Fibroblast Growth Factor 6

Fibroblast Growth Factor 6 (FGF6), also referred to as HST2 or HBGF6, is a member of the Fibroblast Growth Factor (FGF), the Heparin Binding Growth Factor (HBGF) and the Heparin Binding Secretory Transforming Gene (HST) families. The genomic and protein structure of FGF6 is highly conserved among varied species, as is its expression in muscle and muscle progenitor cells. Like other members of the FGF family, FGF6 regulates cell proliferation, differentiation, and migration. Specifically, it plays key roles in myogenesis and muscular regeneration, angiogenesis, along with iron transport and lipid metabolism. Similar to others from the FGF family, FGF6 also possesses oncogenic transforming activity, and as such is implicated in a variety of cancers.

Skeletal muscle vulnerability in a child with Pitt-Hopkins syndrome

BackgroundTCF4 acts as a transcription factor that binds to the immunoglobulin enhancer Mu-E5/KE5 motif. Dominant variants in TCF4 are associated with the manifestation of Pitt-Hopkins syndrome, a rare disease characterized by severe mental retardation, certain features of facial dysmorphism and, in many cases, with abnormalities in respiratory rhythm (episodes of paroxysmal tachypnea and hyperventilation, followed by apnea and cyanosis). Frequently, patients also develop epilepsy, microcephaly, and postnatal short stature. Although TCF4 is expressed in skeletal muscle and TCF4 seems to play a role in myogenesis as demonstrated in mice, potential myopathological findings taking place upon the presence of dominant TCF4 variants are thus far not described in human skeletal muscle.MethodTo address the pathological effect of a novel deletion affecting exons 15 and 16 of TCF4 on skeletal muscle, histological and immunofluorescence studies were carried out on a quadriceps biopsy in addition to targeted transcript studies and global proteomic profiling.ResultsWe report on muscle biopsy findings from a Pitt-Hopkins patient with a novel heterozygous deletion spanning exon 15 and 16 presenting with neuromuscular symptoms. Microscopic characterization of the muscle biopsy revealed moderate fiber type I predominance, imbalance in the proportion of fibroblasts co-expressing Vimentin and CD90, and indicate activation of the complement cascade in TCF4-mutant muscle. Protein dysregulations were unraveled by proteomic profiling. Transcript studies confirmed a mitochondrial vulnerability in muscle and confirmed reduced TCF4 expression.ConclusionOur combined findings, for the first time, unveil myopathological changes as phenotypical association of Pitt-Hopkins syndrome and thus expand the current clinical knowledge of the disease as well as support data obtained on skeletal muscle of a mouse model.

Deletion of TECRL promotes skeletal muscle repair by up-regulating EGR2

Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.

MED23 pathogenic variant: genomic-phenotypic analysis.

The mediator complex subunit 23 (MED23) gene encodes a protein that acts as a tail module mediator complex, a multi-subunit co-activator involved in several cellular activities. MED23 has been shown to have substantial roles in myogenesis and other molecular mechanisms. The functions of MED23 in the neurological system remain unclear and the clinical phenotype is not thoroughly described. Whole exome sequencing was used to identify a novel mutation in the MED23 gene. DNA capture probes using next-generation sequencing-based copy number variation analysis with Illumina array were performed. The clinical, demographic, neuroimaging, and electrophysiological data of the patients were collected, and similarly, the data of all reported cases in the literature were extracted to compare findings. Screening a total of 9,662 articles, we identified 22 main regulatory processes for the MED23 gene, including suppressive activity for carcinogenic processes. MED23 is also involved in the brain's neurogenesis and functions. The identified cases mainly presented with intellectual disability (87.5%) and developmental delay (50%). Seizures were present in only 18.75% of the patients. Slow backgrounds and spike and sharp-wave complexes were reported on the electroencephalogram (EEG) of a few patients and delayed myelination, thin corpus callosum, and pontine hypoplasia on magnetic resonance imaging (MRI). The MED23 gene regulates several processes in which its understanding promotes considerable therapeutic potential for patients. It is crucial to consider genetic and laboratory testing, particularly when encountering potential carriers. Intellectual disability and developmental delay are the most notable clinical signs with heterogeneous features on EEG and MRI.

Fibroblast Growth Factor 6

Fibroblast Growth Factor 6 (FGF6), also referred to as HST2 or HBGF6, is a member of the Fibroblast Growth Factor (FGF), the Heparin Binding Growth Factor (HBGF) and the Heparin Binding Secretory Transforming Gene (HST) families. The genomic and protein structure of FGF6 is highly conserved among varied species, as is its expression in muscle and muscle progenitor cells. Like other members of the FGF family, FGF6 regulates cell proliferation, differentiation, and migration. Specifically, it plays key roles in myogenesis and muscular regeneration, angiogenesis, along with iron transport and lipid metabolism. Similar to others from the FGF family, FGF6 also possesses oncogenic transforming activity, and as such is implicated in a variety of cancers.

Regulation of myo-miR-24-3p on the Myogenesis and Fiber Type Transformation of Skeletal Muscle.

Skeletal muscle plays critical roles in providing a protein source and contributing to meat production. It is well known that microRNAs (miRNAs) exert important effects on various biological processes in muscle, including cell fate determination, muscle fiber morphology, and structure development. However, the role of miRNA in skeletal muscle development remains incompletely understood. In this study, we observed a critical miRNA, miR-24-3p, which exhibited higher expression levels in Tongcheng (obese-type) pigs compared to Landrace (lean-type) pigs. Furthermore, we found that miR-24-3p was highly expressed in the dorsal muscle of pigs and the quadriceps muscle of mice. Functionally, miR-24-3p was found to inhibit proliferation and promote differentiation in muscle cells. Additionally, miR-24-3p was shown to facilitate the conversion of slow muscle fibers to fast muscle fibers and influence the expression of GLUT4, a glucose transporter. Moreover, in a mouse model of skeletal muscle injury, we demonstrated that overexpression of miR-24-3p promoted rapid myogenesis and contributed to skeletal muscle regeneration. Furthermore, miR-24-3p was found to regulate the expression of target genes, including Nek4, Pim1, Nlk, Pskh1, and Mapk14. Collectively, our findings provide evidence that miR-24-3p plays a regulatory role in myogenesis and fiber type conversion. These findings contribute to our understanding of human muscle health and have implications for improving meat production traits in livestock.

Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

CircUBE3C modulates myoblast development by binding to miR-191 and upregulating the expression of p27.

Noncoding RNAs, including miRNAs (microRNAs) and circRNAs (circular RNA), are crucial regulators of myoblast proliferation and differentiation during muscle development. However, the specific roles and molecular mechanisms of circRNAs in muscle development remain poorly understood. Based on the existing circRNA-miRNA-mRNA network, our study focuses on circUBE3C, exploring its differential expression in fetal and adult muscle tissue of the cattle and investigating its impact on myoblast proliferation, apoptosis, and differentiation. The functional analysis of overexpression plasmids and siRNAs (small interfering RNAs) targeting circUBE3C was comprehensively evaluated by employing an array of advanced assays, encompassing CCK-8 (cell counting kit-8), EdU (5-ethynyl-20-deoxyuridine), flow cytometry, western blot analysis, and RT-qPCR. In vivo investigations indicated that overexpression of circUBE3C impedes the process of skeletal muscle regeneration. Mechanistically, we demonstrated that circUBE3C interacts with miR-191 and alleviates the suppression of p27 through cytoplasmic separation, bioinformatics prediction, dual-luciferase reporter assay, and RIP (RNA immunoprecipitation). Our findings indicate that the novel circRNA circUBE3C competitively binds to miR-191, thereby inhibiting proliferation and promoting apoptosis in bovine primary myoblasts and unveiling a regulatory pathway in bovine skeletal muscle development. These findings expand our understanding of circRNA functions in mammals and provide a basis for further exploration of their role in myogenesis and muscle diseases.

Tumor necrosis factor α deficiency promotes myogenesis and muscle regeneration.

Tumor necrosis factor α (TNFα) exhibits diverse biological functions; however, its regulatory roles in myogenesis are not fully understood. In the present study, we explored the function of TNFα in myoblast proliferation, differentiation, migration, and myotube fusion in primary myoblasts and C2C12 cells. To this end, we constructed TNFα muscle-conditional knockout ( TNFα-CKO) mice and compared them with flox mice to assess the effects of TNFα knockout on skeletal muscles. Results indicated that TNFα-CKO mice displayed phenotypes such as accelerated muscle development, enhanced regenerative capacity, and improved exercise endurance compared to flox mice, with no significant differences observed in major visceral organs or skeletal structure. Using label-free proteomic analysis, we found that TNFα-CKO altered the distribution of several muscle development-related proteins, such as Hira, Casz1, Casp7, Arhgap10, Gas1, Diaph1, Map3k20, Cfl2, and Igf2, in the nucleus and cytoplasm. Gene set enrichment analysis (GSEA) further revealed that TNFα deficiency resulted in positive enrichment in oxidative phosphorylation and MyoD targets and negative enrichment in JAK-STAT signaling. These findings suggest that TNFα-CKO positively regulates muscle growth and development, possibly via these newly identified targets and pathways.

Differentiation-dependent chromosomal organization changes in normal myogenic cells are absent in rhabdomyosarcoma cells.

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement. We compared the organizational characteristics of chromosomes 2 and 18, chosen for their difference in myogenic gene arrangement, in cultured RMS cell lines and normal myoblasts and myotubes. We found chromosome-specific differences in organization during normal myogenesis, with increased area occupied and a shift in peripheral localization specifically for chromosome 2. Most strikingly, we found a differentiation-dependent difference in positioning of chromosome 2 relative to the nuclear axis, with preferential positioning along the major nuclear axis present only in myotubes. RMS cells demonstrated no preference for such axial positioning, but induced differentiation through transfection of the pro-myogenic miRNA miR-206 resulted in an increase of major axial positioning of chromosome 2. Our findings identify both a differentiation-dependent, chromosome-specific change in organization in normal myogenesis, and highlight the role of chromosomal spatial organization in myogenic differentiation.

Lactate promotes myogenesis via activating H3K9 lactylation-dependent up-regulation of Neu2 expression.

Lactate, a glycolytic metabolite mainly produced in muscles, has been suggested to regulate myoblast differentiation, although the underlying mechanism remains elusive. Recently, lactate-mediated histone lactylation is identified as a novel epigenetic modification that promotes gene transcription. We used mouse C2C12 cell line and 2-month-old male mice as in vitro and in vivo models, respectively. These models were treated with lactate to explore the biological function and latent mechanism of lactate-derived histone lactylation on myogenic differentiation by quantitative real-time PCR, western blotting, immunofluorescence staining, chromatin immunoprecipitation, cleavage under targets and tagmentation assay and RNA sequencing. Using immunofluorescence staining and western blotting, we proposed that lactylation might occur in the histones. Inhibition of lactate production or intake both impaired myoblast differentiation, accompanied by diminished lactylation in the histones. Using lactylation site-specific antibodies, we demonstrated that lactate preferentially increased H3K9 lactylation (H3K9la) during myoblast differentiation (CT VS 5, 10, 15, 20, 25mM lactate treatment, P=0.0012, P=0.0007, and the rest of all P<0.0001). Notably, inhibiting H3K9la using P300 antagonist could block lactate-induced myogenesis. Through combined omics analysis using cleavage under targets and tagmentation assay and RNA sequencing, we further identified Neu2 as a potential target gene of H3K9la. IGV software analysis (P=0.0013) and chromatin immunoprecipitation-qPCR assay (H3K9la %Input, LA group=9.0076, control group=2.7184, IgG=0.3209) confirmed that H3K9la is enriched in the promoter region of Neu2. Moreover, siRNAs or inhibitors against Neu2 both abrogated myoblast differentiation despite lactate treatment, suggesting that Neu2 is required for lactate-mediated myoblast differentiation. Our findings provide novel understanding of histone lysine lactylation, suggesting its role in myogenesis, and as potential therapeutic targets for muscle diseases.

FLII Modulates the Myogenic Differentiation of Progenitor Cells via Actin Remodeling-Mediated YAP1 Regulation.

The dynamic rearrangement of the actin cytoskeleton plays an essential role in myogenesis, which is regulated by diverse mechanisms, such as mechanotransduction, modulation of the Hippo signaling pathway, control of cell proliferation, and the influence of morphological changes. Despite the recognized importance of actin-binding protein Flightless-1 (FLII) during actin remodeling, the role played by FLII in the differentiation of myogenic progenitor cells has not been explored. Here, we investigated the roles of FLII in the proliferation and differentiation of myoblasts. FLII was found to be enriched in C2C12 myoblasts, and its expression was stable during the early stages of differentiation but down-regulated in fully differentiated myotubes. Knockdown of FLII in C2C12 myoblasts resulted in filamentous actin (F-actin) accumulation and inhibited Yes-associated protein 1 (YAP1) phosphorylation, which triggers its nuclear translocation from the cytoplasm. Consequently, the expressions of YAP1 target genes, including PCNA, CCNB1, and CCND1, were induced, and the cell cycle and proliferation of myoblasts were promoted. Moreover, FLII knockdown significantly inhibited the expression of myogenic regulatory factors, i.e., MyoD and MyoG, thereby impairing myoblast differentiation, fusion, and myotube formation. Thus, our findings demonstrate that FLII is crucial for the differentiation of myoblasts via modulation of the F-actin/YAP1 axis and suggest that FLII is a putative novel therapeutic target for muscle wasting.

CircLRRFIP1 promotes the proliferation and differentiation of chicken skeletal muscle satellite cells by sponging the miR-15 family via activating AKT3-mTOR/p70S6K signaling pathway

Skeletal muscle is important for animal meat production, regulating movements, and maintaining homeostasis. Circular RNAs (circRNAs) have been founded to play vital role in myogenesis. However, the effects of the numerous circRNAs on growth and development of the skeletal muscle are yet to be uncovered. Herein, we identified circLRRFIP1, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. To study the role of circLRRFIP1 in the skeletal muscle, the skeletal muscle satellite cells (SMSCs) was used to silenced or overexpressed circLRRFIP1. The results obtained in this study showed that circLRRFIP1 play a positive role in the proliferation and differentiation of SMSCs. The SMSCs were generated with stable knockdown and overexpression of circLRRFIP1, and the results showed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. We further generated SMSCs with stable knockdown and overexpression of circLRRFIP1, and the results revealed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. Mechanistically, circLRRFIP1 targets the myogenic inhibitory factor-miR-15 family to release the suppression of the miR-15 family to AKT3. The knockdown of AKT inhibits SMSC differentiation through the mTOR/p70S6K pathway. Taken together, the results obtained in this present study revealed the important role and the regulatory mechanisms of circLRRFIP1 in the development of chicken skeletal muscle. Therefore, this study provides an attractive target for molecular breeding to enhance meat production in the chicken industry.

Goat Pleomorphic Adenoma Gene 1 (PLAG1): mRNA Expression, CNV Detection and Associations with Growth Traits.

The pleomorphic adenoma gene 1 (PLAG1) gene, as the major gene responsible for growth, plays a vital role in myogenesis. Meanwhile, the relationship between copy number variation (CNV) of this gene and growth traits in goats remains unclear. Therefore, this study investigated four aspects: bioinformatics analysis, mRNA expression (n = 6), CNV detection (n = 224), and association analysis. The findings indicated that the gene had a large number of conserved motifs, and the gene expression level was higher in fetal goats than in adult goats. Three CNV loci were selected from the database, among which CNV1 was located in the bidirectional promoter region and was associated with goat growth traits. CNV analysis showed that CNV2 and CNV3 of the PLAG1 gene were associated with growth traits such as body weight, heart girth, height at hip cross, and hip width (p < 0.05), with CNV1 loss genotype being the superior genotype, and CNV2 and CNV3 median and gain genotypes of being superior genotypes. This finding further confirms that the PLAG1 gene is the dominant gene for growth traits, which will serve as theoretical guidance for goat breeding.

Saturated fatty acid-inducible miR-103-3p impairs the myogenic differentiation of progenitor cells by enhancing cell proliferation through Twinfilin-1/F-actin/YAP1 axis

Actin dynamics play an essential role in myogenesis through multiple mechanisms, such as mechanotransduction, cell proliferation, and myogenic differentiation. Twinfilin-1 (TWF1), an actin-depolymerizing protein, is known to be required for the myogenic differentiation of progenitor cells. However, the mechanisms by which they epigenetically regulate TWF1 by microRNAs under muscle wasting conditions related to obesity are almost unknown. Here, we investigated the role of miR-103-3p in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation of progenitor cells. Palmitic acid, the most abundant saturated fatty acid (SFA) in the diet, reduced TWF1 expression and impeded myogenic differentiation of C2C12 myoblasts, while elevating miR-103-3p levels in myoblasts. Interestingly, miR-103-3p inhibited TWF1 expression by directly targeting its 3'UTR. Furthermore, ectopic expression of miR-103-3p reduced the expression of myogenic factors, i.e., MyoD and MyoG, and subsequently impaired myoblast differentiation. We demonstrated that miR-103-3p induction increased filamentous actin (F-actin) and facilitated the nuclear translocation of Yes-associated protein 1 (YAP1), thereby stimulating cell cycle progression and cell proliferation. Hence, this study suggests that epigenetic suppression of TWF1 by SFA-inducible miR-103-3p impairs myogenesis by enhancing the cell proliferation triggered by F-actin/YAP1.

Transcriptomic Analysis Reveals mRNA and Alternative Splicing Events in Ovine Skeletal Muscle Satellite Cells during Proliferation and Differentiation

Skeletal muscle satellite cells (SMSCs), which are highly multifunctional muscle-derived stem cells, play an essential role in myogenesis and regeneration. Here, the transcriptional profile of SMSCs during proliferation and differentiation were constructed using the RNA-Seq method. A total of 1954 differentially expressed genes (DEGs) and 1092 differentially alternative splicing genes (DAGs) were identified including 1288 upregulated genes as well as 666 downregulated genes. GO and KEGG analyses showed that the DEGs and DAGs were enriched in the MAPK (mitogen-activated protein kinase) signaling pathway, the PI3K-Akt (phosphatidylinositol-tris-phosphate kinase 3/protein kinase B) signaling pathway, the Wnt signaling pathway, and the Ras signaling pathway. In total, 1479 alternative splice events (AS) were also identified during SMSC proliferation and differentiation. Among them, a unique AS event was the major per-mRNA splicing type, and SE was the predominant splicing pattern. Furthermore, transcription factors with AS were scanned during SMSC differentiation such as myocyte enhancer factor-2C (MEF2C) and the nuclear receptor subfamily 4 group A member 2 (NR4A2). Our results imply that MEF2C and NR4A2 can interact, and we speculate that NR4A2 and MEF2C might regulate the myogenesis of ovine SMSCs through interaction. Together, our study provides useful information on the transcriptional regulation of SMSCs during proliferation and differentiation at the transcriptional level, and provides a valuable resource for understanding the molecular mechanism of myogenesis and muscle development.

GNRH family genes contributed to gender-specific disparity of bladder cancer prognosis through exerting opposite regulatory roles between males and females.

There is gender-specific disparity in bladder cancer (BlCa) prognosis. Female BlCa patients present with more advanced tumor and have higher risks of disease recurrence, progression, and mortality than males. Since gonadotropin-releasing hormone (GNRH) family genes were critical genes in gender-related biological activity and could be detected in BlCa specimens, this study aimed to explore potential roles of GNRH1 and GNRHR in gender disparity of BlCa. RNA-sequencing data from The Cancer Genome Atlas Bladder Urothelial Carcinoma dataset, IMvigor210 immunotherapy cohort and Cancer Cell Line Encyclopedia database were used to compare potential roles of GNRH1 and GNRHR in males and females, respectively. Gene set enrichment analysis was used to analyze the biological functions. Males with higher GNRH1 and GNRHR have better overall survival (P < 0.05, HR < 1), while females with higher expression have a trend toward worse overall survival (P < 0.05, HR > 1). Gene set enrichment analysis identified GNRH1 and GNRHR exert opposite regulatory roles in myogenesis (M5909), interferon-α response (M5911), interferon-γ response (M5913), inflammatory response (M5932) and TNF-α signaling via NF-κβ (M5890) between males and females. The five functions are up-regulated in females (NES > 0), while down-regulated in males (NES < 0). GNRH1 in females was positively correlated with CD3D (R-value > 0 and P < 0.05), while GNRHR in males was negatively correlated with CD247, CD3D and CD3E (R-value < 0 and P < 0.05). GNRH1 and GNRHR have opposite effects on overall survival in different genders, and exert opposite roles in immune-related functions between different genders, which could emerge as a contributor to gender disparity of BlCa prognosis.