Gene expression profiles of specific chicken skeletal muscles.

The chicken provides large amounts of protein for the human diet and is also used as a model organism for biomedical research. Increasing meat production is an important goal in the poultry industry and skeletal muscles have highly diverse origins, shapes, metabolic features, and physical functions. Previous gene expression atlases have largely ignored the differences among diverse types of skeletal muscles; therefore, comprehensive transcriptional maps of all skeletal muscles are needed to improve meat production traits. In this study, we sequenced 58 samples from 10 different skeletal muscles of 42-day-old White Plymouth Rock chickens. We also measured myofiber diameter and generated myofiber-type datasets of these 10 tissues. We generated 418.4 Gb high-quality bulk RNA-Seq data from four or six biological replicates of each skeletal muscle (four replicates from extraocular samples) (approximately 7.4 Gb per sample). This dataset provides valuable information for understanding the muscle fiber characteristics of White Plymouth Rock chickens. Furthermore, our data can be used as a model for heterogeneity analysis between tissues with similar properties.

Identification and expression pattern analysis of miRNAs in pectoral muscle during pigeon (Columba livia) development.

MicroRNAs (miRNAs) are a group of crucial regulators in the process of animal growth and development. However, little is known about the expression and function of miRNAs in pigeon muscles. To identify the miRNAs participating in the rapid development of pigeon pectoral muscles and quantitate their expression levels of pectoral muscles in different age stages, we performed miRNA transcriptome analysis in pigeon pectoral muscles by sequencing small RNAs over three different age stages (1-day old, 28 days old, and 2 years old). Dual-luciferase reporter assay was applied to validate the interaction between miRNA and its target gene. We identified 304 known miRNAs, 201 conserved miRNAs, and 86 novel miRNAs in pigeon pectoral muscles. 189 differentially expressed (DE) miRNAs were screened out during pigeon development. A short time-series expression miner (STEM) analysis indicated 89 DE miRNAs were significantly clustered in a progressively decreasing expression profile, and mainly enriched in biosynthesis-related GO categories and signaling pathways for MAPK and TGF-ß. Dual-luciferase reporter assay indicated that a progressively down-regulated miRNA (miR-20b-5p) could directly target Krüppel-like factor 3 (KLF3) gene. To sum-up, our data expand the repertoire of pigeon miRNAs and enhance understanding of the mechanisms underlying rapid development in squabs.

Dietary betaine prevents obesity through gut microbiota-drived microRNA-378a family.

Betaine is a natural compound present in commonly consumed foods and may have a potential role in the regulation of glucose and lipids metabolism. However, the underlying molecular mechanism of its action remains largely unknown. Here, we show that supplementation with betaine contributes to improved high-fat diet (HFD)-induced gut microbiota dysbiosis and increases anti-obesity strains such as Akkermansia muciniphila, Lactobacillus, and Bifidobacterium. In mice lacking gut microbiota, the functional role of betaine in preventing HFD-induced obesity, metabolic syndrome, and inactivation of brown adipose tissues are significantly reduced. Akkermansia muciniphila is an important regulator of betaine in improving microbiome ecology and increasing strains that produce short-chain fatty acids (SCFAs). Increasing two main members of SCFAs including acetate and butyrate can significantly regulate the levels of DNA methylation at host miR-378a promoter, thus preventing the development of obesity and glucose intolerance. However, these beneficial effects are partially abolished by Yin yang (YY1), a common target gene of the miR-378a family. Taken together, our findings demonstrate that betaine can improve obesity and associated MS via the gut microbiota-derived miR-378a/YY1 regulatory axis, and reveal a novel mechanism by which gut microbiota improve host health.

Comprehensive Analysis of mRNA and lncRNA Transcriptomes Reveals the Differentially Hypoxic Response of Preadipocytes During Adipogenesis.

Local hypoxia has recently been reported to occur in the white adipose tissue (WAT) microenvironment during obesity. Adipocytes have a unique life cycle that reflects the different stages of adipogenesis in the WAT niche. Long non-coding RNAs (lncRNAs) play an important role in the cellular response to hypoxia. However, the differentially hypoxic responses of preadipocytes during adipogenesis and the potential role of lncRNAs in this process remain to be elucidated. Here, we evaluated the differentially hypoxic responses of primary hamster preadipocytes during adipogenesis and analyzed mRNA and lncRNA expression in same Ribo-Zero RNA-seq libraries. Hypoxia induced HIF-1α protein during adipogenesis and caused divergent changes of cell phenotypes. A total of 10,318 mRNAs were identified to be expressed in twenty libraries (five timepoints), and 3,198 differentially expressed mRNAs (DE mRNAs) were detected at five timepoints (hypoxia vs. normoxia). Functional enrichment analysis revealed the shared and specific hypoxia response pathways in the different stages of adipogenesis. Hypoxia differentially modulated the expression profile of adipose-associated genes, including adipokines, lipogenesis, lipolysis, hyperplasia, hypertrophy, inflammatory, and extracellular matrix. We also identified 4,296 lncRNAs that were expressed substantially and detected 1,431 DE lncRNAs at five timepoints. Two, 3, 5, 13, and 50 DE mRNAs at D0, D1, D3, D7, and D11, respectively, were highly correlated and locus-nearby DE lncRNAs and mainly involved in the cell cycle, vesicle-mediated transport, and mitochondrion organization. We identified 28 one-to-one lncRNA-mRNA pairs that might be closely related to adipocyte functions, such as ENSCGRT00015041780-Hilpda, TU2105-Cdsn, and TU17588-Ltbp3. These lncRNAs may represent the crucial regulation axis in the cellular response to hypoxia during adipogenesis. This study dissected the effects of hypoxia in the cell during adipogenesis, uncovered novel regulators potentially associated with WAT function, and may provide a new viewpoint for interpretation and treatment of obesity.

Comprehensive Analysis of lncRNAs and circRNAs Reveals the Metabolic Specialization in Oxidative and Glycolytic Skeletal Muscles.

The biochemical and functional differences between oxidative and glycolytic muscles could affect human muscle health and animal meat quality. However, present understanding of the epigenetic regulation with respect to lncRNAs and circRNAs is rudimentary. Here, porcine oxidative and glycolytic skeletal muscles, which were at the growth curve inflection point, were sampled to survey variant global expression of lncRNAs and circRNAs using RNA-seq. A total of 4046 lncRNAs were identified, including 911 differentially expressed lncRNAs (p < 0.05). The cis-regulatory analysis identified target genes that were enriched for specific GO terms and pathways (p < 0.05), including the oxidation-reduction process, glycolytic process, and fatty acid metabolic. All these were closely related to different phenotypes between oxidative and glycolytic muscles. Additionally, 810 circRNAs were identified, of which 137 were differentially expressed (p < 0.05). Interestingly, some circRNA-miRNA-mRNA networks were found, which were closely linked to muscle fiber-type switching and mitochondria biogenesis in muscles. Furthermore, 44.69%, 39.19%, and 54.01% of differentially expressed mRNAs, lncRNAs, and circRNAs respectively were significantly enriched in pig quantitative trait loci (QTL) regions for growth and meat quality traits. This study reveals a mass of candidate lncRNAs and circRNAs involved in muscle physiological functions, which may improve understanding of muscle metabolism and development from an epigenetic perspective.

miR-27a-5p Attenuates Hypoxia-induced Rat Cardiomyocyte Injury by Inhibiting Atg7.

Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality worldwide. AMI triggers a hypoxic microenvironment and induces extensive myocardial injury, including autophagy and apoptosis. MiRNAs, which are a class of posttranscriptional regulators, have been shown to be involved in the development of ischemic heart diseases. We have previously reported that hypoxia significantly alters the miRNA transcriptome in rat cardiomyoblast cells (H9c2), including miR-27a-5p. In the present study, we further investigated the potential function of miR-27a-5p in the cardiomyocyte response to hypoxia, and showed that miR-27a-5p expression was downregulated in the H9c2 cells at different hypoxia-exposed timepoints and the myocardium of a rat AMI model. Follow-up experiments revealed that miR-27a-5p attenuated hypoxia-induced cardiomyocyte injury by regulating autophagy and apoptosis via Atg7, which partly elucidated the anti-hypoxic injury effects of miR-27a-5p. Taken together, this study shows that miR-27a-5p has a cardioprotective effect on hypoxia-induced H9c2 cell injury, suggesting it may be a novel target for the treatment of hypoxia-related heart diseases.

Genistein reverses isoproterenol-induced cardiac hypertrophy by regulating miR-451/TIMP2.

Cardiomyocyte hypertrophy, a prevalent clinical condition is deeply associated with many physiological factors. The underlying mechanisms of cardiomyocyte hypertrophy are not yet fully understood. In this study, H9C2 cells were treated with genistein, miR-451 mimic, miR-451 inhibitor and isoproterenol for 24 h, to study the effect of genistein on isoproterenol-induced myocardial hypertrophy in vitro. Simultaneously, ICR mice were treated with genistein for 21 days to evaluate the effects of the phytochemical on isoproterenol-induced myocardial hypertrophy in vivo. Results showed that isoproterenol induced cardiac hypertrophy and down-regulated the expression of miR-451 and up-regulated miR-451's target gene TIMP2. Genistein increased the expression of miR-451 and inhibited the isoproterenol-induced cardiac hypertrophy. This study explored the function of genistein from the epigenetic level, suggesting that miR-451 may play a significant role in the genistein-assisted amelioration of isoproterenol-induced cardiac hypertrophy both in vitro and in vivo.

MicroRNA-143a-3p modulates preadipocyte proliferation and differentiation by targeting MAPK7.

Adipogenesis plays a key role in increasing fat mass, which is a main characteristic for obesity, and involves preadipocyte proliferation and differentiation. Recently, more and more evidences suggested that microRNAs (miRNAs) is an important member of the regulatory network of adipogenesis. In this study, miR-143a-3p was highly expressed in adipose tissues of obese mice, and was up-regulated at the middle and last stage of 3T3-L1 adipocyte differentiation. Using mouse 3T3-L1 cells line, which is an ideal model in vitro for the study of adipogenesis, we observed that overexpression of miR-143a-3p inhibited the preadipocyte proliferation, and enhanced the preadipocyte differentiation. In contrast, the inhibition of miR-143a-3p expression promoted the preadipocyte proliferation, and inhibited the preadipocyte differentiation. Further analysis suggested that miR-143a-3p mediating preadipocyte differentiation might be involved in fatty acid metabolism. In addition, we found that miR-143-3p and PPARγ, an activator of miR-143a-3p transcription, could regulate each other. Compared with miR-143a-3p, MAPK7 played an opposite role in the proliferation and differentiation of adipocyte. Further analysis indicated that MAPK7 is a target gene of miR-143a-3p in 3T3-L1 cells, and inhibition of MAPK7 recede the effect of miR-143a-3p on preadipocyte proliferation and differentiation. Taken together, these results indicated that as a regulator of PPARγ, miR-143a-3p play an important role in adipogenesis via regulating MAPK7 and fatty acid.

Global Long Noncoding RNA and mRNA Expression Changes between Prenatal and Neonatal Lung Tissue in Pigs.

Lung tissue plays an important role in the respiratory system of mammals after birth. Early lung development includes six key stages, of which the saccular stage spans the pre- and neonatal periods and prepares the distal lung for alveolarization and gas-exchange. However, little is known about the changes in gene expression between fetal and neonatal lungs. In this study, we performed transcriptomic analysis of messenger RNA (mRNA) and long noncoding RNA (lncRNA) expressed in the lung tissue of fetal and neonatal piglets. A total of 19,310 lncRNAs and 14,579 mRNAs were identified and substantially expressed. Furthermore, 3248 mRNAs were significantly (FDR-adjusted p value ≤ 0.05, FDR: False Discovery Rate) differentially expressed and were mainly enriched in categories related to cell proliferation, immune response, hypoxia response, and mitochondrial activation. For example, CCNA2, an important gene involved in the cell cycle and DNA replication, was upregulated in neonatal lungs. We also identified 452 significantly (FDR-adjusted p value ≤ 0.05) differentially expressed lncRNAs, which might function in cell proliferation, mitochondrial activation, and immune response, similar to the differentially expressed mRNAs. These results suggest that differentially expressed mRNAs and lncRNAs might co-regulate lung development in early postnatal pigs. Notably, the TU64359 lncRNA might promote distal lung development by up-regulating the heparin-binding epidermal growth factor-like (HB-EGF) expression. Our research provides basic lung development datasets and will accelerate clinical researches of newborn lung diseases with pig models.

Long Noncoding RNA GAS5 Suppresses 3T3-L1 Cells Adipogenesis Through miR-21a-5p/PTEN Signal Pathway.

Emerging studies indicated that both long noncoding RNAs and micro-RNAs play crucial roles in the mediation of adipogenesis, which is closely linked to obesity-related diseases. However, the mechanisms of lncRNA-miRNAs coregulating in adipogenesis are still largely unknown. In this study, we determined that lncRNA growth arrest-specific 5 (GAS5) presented an opposite expression pattern with miR-21a-5p in 3T3-L1 adipocytes development. To explore the role of GAS5 in adipogenesis, pcDNA3.1-GAS5 expression vectors and GAS5-siRNAs were used to perform GAS5 overexpression and knockdown, respectively. Ectopic expression of GAS5 dramatically reduced miR-21a-5p level and suppressed the proliferation of 3T3-L1 preadipocytes, while silencing GAS5 slightly increased miR-21a-5p expression but had no significant influence on the cell viability. In addition, overexpression of GAS5 remarkably decreased the mRNA and protein levels of adipogenic marker genes, and resulted in a notable reduction of lipid accumulation. In contrast, overexpressing miR-21a-5p significantly facilitated differentiation of 3T3-L1 cells. By target gene prediction and luciferase reporter assay, we suggested that GAS5 might indirectly improve the expression of phosphatase and tensin homolog (PTEN) by repressing miR-21a-5p in a miRNA-based regulatory mechanism. Together, GAS5 plays a suppressive role in 3T3-L1 cells adipogenesis, which further highlights the importance of lncRNAs in adipogenesis.

The regulation of skeletal muscle fiber-type composition by betaine is associated with NFATc1/MyoD.

Increasing evidence indicates that muscular dysfunction or alterations in skeletal muscle fiber-type composition not only are involved in muscle metabolism and function but also can limit functional capacity. Therefore, understanding the mechanisms regulating key events during skeletal myogenesis is necessary. Betaine is a naturally occurring component of commonly eaten foods. Here, we showed that 10 mM betaine supplementation in vitro significantly repressed myoblast proliferation and enhanced myoblast differentiation. This effect can be mediated by regulation of miR-29b-3p. Further analysis showed that betaine supplementation in vitro regulated skeletal muscle fiber-type composition through the induction of NFATc1 and the negative regulation of MyoD expression. Furthermore, mice fed with 10 mM betaine in water for 133 days showed no impairment in overall health. Consistently, betaine supplementation increased muscle mass, promoted muscle formation, and modulated the ratio of fiber types in skeletal muscle in vivo. These findings shed light on the diverse biological functions of betaine and indicate that betaine supplementation may lead to new therapies for diseases such as muscular dystrophy or other diseases related to muscle dysfunction. KEY MESSAGES: Betaine supplementation inhibits proliferation and promotes differentiation of C2C12 myoblasts. Betaine supplementation regulates fast to slow muscle fiber-type conversion and is associated with NFATc1/MyoD. Betaine supplementation enhances skeletal myogenesis in vivo. Betaine supplementation does not impair health of mice.

Spontaneous single nucleotide polymorphism in porcine microRNA-378 seed region leads to functional alteration.

Sequence variation in a microRNA (miRNA) seed region can influence its biogenesis and effects on target mRNAs; however, in mammals, few seed region mutations leading to functional alterations have been reported to date. Here, we report the identification of a single nucleotide polymorphism (SNP) with functional consequence located in the seed region of porcine miR-378. In vitro analysis of this rs331295049 A17G SNP showed significantly up-regulated expression of the mature miR-378 (miR-378/G). In silico target prediction indicated that the SNP would modulate secondary structure and result in functional loss affecting >85% of the known target genes of the wild-type miR-378 (miR-378/A), and functional gain affecting >700 new target genes, and dual-luciferase reporter assay verified this result. This report of a SNP in the seed region of miR-378 leads to functional alteration and indicates the potential for substantive functional consequences to the molecular physiology of a mammalian organism.

miR-144-3p Promotes Adipogenesis Through Releasing C/EBPα From Klf3 and CtBP2.

MicroRNAs (miRNAs), a class of small non-coding RNAs, have been proved as novel and potent regulators of adipogenesis. A previous study has found out that miR-144-3p was a biomarker of type 2 diabetes, but the role of miR-144-3p in regulating adipogenesis was still unclear. In the present study, the expression of miR-144-3p increased in obese mice and during the 3T3-L1 differentiation process. Overexpression of miR-144-3p suppressed the expression of cell cycle regulatory factors and inhibited pre-adipocytes proliferation. Besides, overexpression of miR-144-3p accelerated lipid accumulation in adipocytes and positively regulated adipogenesis, which was also accompanied by increasing the expression of genes related to fatty acid synthesis and decreasing the expression of genes involved in fatty acid oxidation. Furthermore, luciferase activity assays indicated that miR-144-3p directly targeted Klf3 and CtBP2. The process was also confirmed by the mRNA and protein expression of Klf3 and CtBP2, which were suppressed by miR-144-3p. Furthermore, miR-144-3p targeting Klf3/CtBP2 would induce C/EBPα activity by releasing corepressors (Klf3 and CtBP2) from its promoter region. Moreover, we also observed that miR-144-3p could promote adipogenesis in mice injected with miR-144-3p agomir through tail-vein injection. Taken together, these results support that miR-144-3p can facilitate adipogenesis both in vitro and in vivo, which implies that miR-144-3p could be a target for therapeutic intervention in obesity and metabolic syndrome in the future.

High-Altitude Living Shapes the Skin Microbiome in Humans and Pigs.

While the skin microbiome has been shown to play important roles in health and disease in several species, the effects of altitude on the skin microbiome and how high-altitude skin microbiomes may be associated with health and disease states remains largely unknown. Using 16S rRNA marker gene sequencing, we characterized the skin microbiomes of people from two racial groups (the Tibetans and the Hans) and of three local pig breeds (Tibetan pig, Rongchang pig, and Qingyu pig) at high and low altitudes. The skin microbial communities of low-altitude pigs and humans were distinct from those of high-altitude pigs and humans, with five bacterial taxa (Arthrobacter, Paenibacillus, Carnobacterium, and two unclassified genera in families Cellulomonadaceae and Xanthomonadaceae) consistently enriched in both pigs and humans at high altitude. Alpha diversity was also significantly lower in skin samples collected from individuals living at high altitude compared to individuals at low altitude. Several of the taxa unique to high-altitude humans and pigs are known extremophiles adapted to harsh environments such as those found at high altitude. Altogether our data reveal that altitude has a significant effect on the skin microbiome of pigs and humans.

Transcriptome-wide N 6 -methyladenosine methylome profiling of porcine muscle and adipose tissues reveals a potential mechanism for transcriptional regulation and differential methylation pattern.

BACKGROUND: N 6 -methyladenosine (m6A) is the most prevalent internal form of modification in messenger RNA in higher eukaryotes and potential regulatory functions of reversible m6A methylation on mRNA have been revealed by mapping of m6A methylomes in several species. m6A modification in active gene regulation manifests itself as altered methylation profiles in a tissue-specific manner or in response to changing cellular or species living environment. However, up to date, there has no data on m6A porcine transcriptome-wide map and its potential biological roles in adipose deposition and muscle growth. METHODS: In this work, we used methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) technique to acquire the first ever m6A porcine transcriptome-wide map. Transcriptomes of muscle and adipose tissues from three different pig breeds, the wild boar, Landrace, and Rongchang pig, were used to generate these maps. RESULTS: Our findings show that there were 5,872 and 2,826 m6A peaks respectively, in the porcine muscle and adipose tissue transcriptomes. Stop codons, 3'-untranslated regions, and coding regions were found to be mainly enriched for m6A peaks. Gene ontology analysis revealed that common m6A peaks in nuclear genes are associated with transcriptional factors, suggestive of a relationship between m6A mRNA methylation and nuclear genome transcription. Some genes showed tissue- and breed-differential methylation, and have novel biological functions. We also found a relationship between the m6A methylation extent and the transcript level, suggesting a regulatory role for m6A in gene expression. CONCLUSION: This comprehensive map provides a solid basis for the determination of potential functional roles for RNA m6A modification in adipose deposition and muscle growth.

Coat colour phenotype of Qingyu pig is associated with polymorphisms of melanocortin receptor 1 gene.

OBJECTIVE: Qingyu pig, a Chinese indigenous pig breed, exhibits two types of coat colour phenotypes, including pure black and white with black spotting respectively. Melanocortin receptor 1 (MC1R) and agouti signaling protein (ASIP) are two widely reported pivotal genes that significantly affect the regulation of coat colour. The objectives of this study were to investigate whether the polymorphisms of these two genes are associated with coat colour and analyze the molecular mechanism of the coat colour separation in Qingyu pig. METHODS: We studied the phenotype segregation and used polymerase chain reaction amplification and Sanger sequencing to investigate the polymorphism of MC1R and ASIP in 121 Qingyu pigs, consisting of 115 black and 6 white with black spotted pigs. RESULTS: Coat colour of Qingyu pig is associated with the polymorphisms of MC1R but not ASIP. We only found 2 haplotypes, EQY and Eqy , based on the 13 observed mutations from MC1R gene. Among which, Eqy presented a recessive inheritance mode in black spotted Qingyu pigs. Further analysis revealed a g.462-463CC insertion that caused a frameshift mutation and a premature stop codon, thus changed the first transmembrane domain completely and lost the remaining six transmembrane domains. Altogether, our results strongly support that the variety of Qingyu pig's coat colour is related to MC1R. CONCLUSION: Our findings indicated that black coat colour in Qingyu pig was dominant to white with black spotted phenotype and MC1R gene polymorphism was associated with coat colour separation in Qingyu pig.

Comprehensive variation discovery and recovery of missing sequence in the pig genome using multiple de novo assemblies.

Uncovering genetic variation through resequencing is limited by the fact that only sequences with similarity to the reference genome are examined. Reference genomes are often incomplete and cannot represent the full range of genetic diversity as a result of geographical divergence and independent demographic events. To more comprehensively characterize genetic variation of pigs (Sus scrofa), we generated de novo assemblies of nine geographically and phenotypically representative pigs from Eurasia. By comparing them to the reference pig assembly, we uncovered a substantial number of novel SNPs and structural variants, as well as 137.02-Mb sequences harboring 1737 protein-coding genes that were absent in the reference assembly, revealing variants left by selection. Our results illustrate the power of whole-genome de novo sequencing relative to resequencing and provide valuable genetic resources that enable effective use of pigs in both agricultural production and biomedical research.

Role of miR-181a-5p and endoplasmic reticulum stress in the regulation of myogenic differentiation.

Accumulating evidence has indicated that microRNAs (miRNAs) and endoplasmic reticulum (ER) stress play critical roles in myoblast differentiation. However, the regulation roles of miRNAs and ER stress in myogenic differentiation have not been fully revealed and need to be further studied. Here, we discovered that the expression levels of miR-181a-5p were strongly upregulated during C2C12 cell differentiation. miR-181a-5p overexpression promoted ER stress and differentiation of C2C12 cells, which was accompanied by increasing expression levels of marker genes related to ER stress-mediated apoptosis and myogenic differentiation. Opposite results were observed after inhibition of the miR-181a-5p expression. The gain- and loss-of-function experiments on C2C12 cells showed that miR-181a-5p affected the development of muscle fiber type, but had no significant influence on C2C12 cell proliferation. In the ER-stressed C2C12 cells induced by thapsigargin (Tg), the expression levels of both miR-181a-5p and marker genes related to ER stress and myogenesis were upregulated. In the ER-stressed C2C12 cells and porcine muscle fibroblast (PMF) cells pretreated with Tg, we found that miR-181a-5p targeted glucose-regulated protein, 78kDa/binding immunoglobulin protein (GRP78/BIP), and influenced cell apoptosis. In conclusion, these results indicate that miR-181a-5p and ER stress have positive synergistic effects on myogenic differentiation by increasing the expression levels of myogenic differentiation key genes and activating the ER stress-mediated apoptosis signaling pathway.

miR-145a-5p Promotes Myoblast Differentiation.

MicroRNAs are a class of 18-22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and ß-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation.

Effect of miR-143-3p on C2C12 myoblast differentiation.

MicroRNAs are a class of 18-22 nucleotide non-coding RNAs that modulate gene expression by associating with the 3' untranslated regions of mRNAs. A large number of microRNAs are involved in the regulation of myoblast differentiation, many of which remain undiscovered. In this study, we found that miR-143-3p was upregulated during C2C12 myoblast differentiation and over-expression of miR-143-3p significantly inhibited the relative expression levels of MyoD, MyoG, myf5, and MyHC genes, especially in the later stages of differentiation. In addition, miR-143-3p inhibited expression of genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and ß-catenin. These results indicate that miR-143-3p represents a new myogenic differentiation-associated microRNA that can inhibit C2C12 myoblast differentiation, especially in the later stages of differentiation.