Effects of active immunization against porcine Sox6 on meat quality and myosin heavy chain isoform expression in growing-finishing pigs

A feeding trial for 91 days was conducted to investigate effects of active immunization against porcine Sox6 (pSox6) on meat quality and myosin heavy chain (MyHC) isoform expression in growing-finishing pigs. Twenty-four castrated Duroc?×?Landrace?×?Yarkshire pigs were randomly divided into three groups: (1) Control group; (2) 1?mg/head pSox6 active immunity group; (3) 4?mg/head pSox6 active immunity group (4?mg/head group). The results showed that pigs in 4?mg/head group had a greater a* (Redness) and a higher marbling score, while no significant effect was observed in L* (Lightness), b* (Yellowness), intramuscular fat and cooking loss. Muscle succinic dehydrogenase activity in pSox6 active immunization groups was significantly increased, and muscle lactate dehydrogenase activity was significantly reduced. Meanwhile, active immunization against pSox6 upregulated the mRNA expression of MyHC I, while no effect was observed on the mRNA expressions of MyHC IIa, MyHC IIx, MyHC IIb. In addition, pigs in the 4?mg/head group exhibited lower Sox6 mRNA level and higher MyHC I protein level, while no significant influence was observed on MyHC IIb protein level. Together, our data imply that active immunization against pSox6 could improve the pork quality and promote the MyHC I expression in growing-finishing pigs.     

MicroRNA-499 Expression Distinctively Correlates to Target Genes sox6 and rod1 Profiles to Resolve the Skeletal Muscle Phenotype in Nile Tilapia

A class of small non-coding RNAs, the microRNAs (miRNAs), has been shown to be essential for the regulation of specific cell pathways, including skeletal muscle development, maintenance and homeostasis in vertebrates. However, the relative contribution of miRNAs for determining the red and white muscle cell phenotypes is far from being fully comprehended. To better characterize the role of miRNA in skeletal muscle cell biology, we investigated muscle-specific miRNA (myomiR) signatures in Nile tilapia fish. Quantitative (RT-qPCR) and spatial (FISH) expression analyses revealed a highly differential expression (forty-four-fold) of miR-499 in red skeletal muscle compared to white skeletal muscle, whereas the remaining known myomiRs were equally expressed in both muscle cell types. Detailed examination of the miR-499 targets through bioinformatics led us to the sox6 and rod1 genes, which had low expression in red muscle cells according to RT-qPCR, FISH, and protein immunofluorescence profiling experiments. Interestingly, we verified that the high expression of miR-499 perfectly correlates with a low expression of sox6 and rod1 target genes, as verified by a distinctive predominance of mRNA destabilization and protein translational decay to these genes, respectively. Through a genome-wide comparative analysis of SOX6 and ROD1 protein domains and through an in silico gene regulatory network, we also demonstrate that both proteins are essentially similar in vertebrate genomes, suggesting their gene regulatory network may also be widely conserved. Overall, our data shed light on the potential regulation of targets by miR-499 associated with the slow-twitch muscle fiber type phenotype. Additionally the results provide novel insights into the evolutionary dynamics of miRNA and target genes enrolled in a putative constrained molecular pathway in the skeletal muscle cells of vertebrates.     

Differential microRNA Expression in Fast- and Slow-Twitch Skeletal Muscle of Piaractus mesopotamicus during Growth

Pacu (Piaractus mesopotamicus) is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs), represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4), srf (serum response factor), pax7 (paired box 7) and sox6 ((sex determining region Y)-box 6), respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days), juvenile (aged 90 days and 150 days) and adult (aged 2 years). To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.     

miR-30 Family microRNAs Regulate Myogenic Differentiation and Provide Negative Feedback on the microRNA Pathway

microRNAs (miRNAs) are short non-coding RNAs that can mediate changes in gene expression and are required for the formation of skeletal muscle (myogenesis). With the goal of identifying novel miRNA biomarkers of muscle disease, we profiled miRNA expression using miRNA-seq in the gastrocnemius muscles of dystrophic mdx4cv mice. After identifying a down-regulation of the miR-30 family (miR-30a-5p, -30b, -30c, -30d and -30e) when compared to C57Bl/6 (WT) mice, we found that overexpression of miR-30 family miRNAs promotes differentiation, while inhibition restricts differentiation of myoblasts in vitro. Additionally, miR-30 family miRNAs are coordinately down-regulated during in vivo models of muscle injury (barium chloride injection) and muscle disuse atrophy (hindlimb suspension). Using bioinformatics tools and in vitro studies, we identified and validated Smarcd2, Snai2 and Tnrc6a as miR-30 family targets. Interestingly, we show that by targeting Tnrc6a, miR-30 family miRNAs negatively regulate the miRNA pathway and modulate both the activity of muscle-specific miR-206 and the levels of protein synthesis. These findings indicate that the miR-30 family may be an interesting biomarker of perturbed muscle homeostasis and muscle disease.     

MicroRNA Transcriptome Profile Analysis in Porcine Muscle and the Effect of miR-143 on the MYH7 Gene and Protein

Porcine skeletal muscle fibres are classified based on their different physiological and biochemical properties. Muscle fibre phenotype is regulated by several independent signalling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT), myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) signalling pathways. MicroRNAs are non-coding small RNAs that regulate many biological processes. However, their function in muscle fibre type regulation remains unclear. The aim of our study was to identify miRNAs that regulate muscle fibre type during porcine growth to help understand the miRNA regulation mechanism of fibre differentiation. We performed Solexa/Illumina deep sequencing for the microRNAome during 3 muscle growth stages (63, 98 and 161 d). In this study, 271 mature miRNAs and 243 pre-miRNAs were identified. We detected 472 novel miRNAs in the muscle samples. Among the mature miRNAs, there are 23 highest expression miRNAs (over 10000 RPM), account for 85.3% of the total counts of mature miRNAs., including 10 (43.5%) muscle-related miRNAs (ssc-miR-133a-3p, ssc-miR-486, ssc-miR-1, ssc-miR-143-3p, ssc-miR-30a-5p, ssc-miR-181a, ssc-miR-148a-3p, ssc-miR-92a, ssc-miR-21, ssc-miR-126-5p). Particularly, both ssc-miR-1 and ssc-miR-133 belong to the MyomiRs, which control muscle myosin content, myofibre identity and muscle performance. The involvement of these miRNAs in muscle fibre phenotype provides new insight into the mechanism of muscle fibre regulation underlying muscle development. Furthermore, we performed cell transfection experiment. Overexpression/inhibition of ssc-miR-143-3p in porcine skeletal muscle satellite cell induced an/a increase/reduction of the slow muscle fibre gene and protein (MYH7), indicating that miR-143 activity regulated muscle fibre differentiate in skeletal muscle. And it regulate MYH7 through the HDAC4-MEF2 pathway.     

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.     

Inhibition of miR-214 expression represses proliferation and differentiation of C2C12 myoblasts

MicroRNAs (miRNAs) are small non-coding RNAs that participate in diverse biological processes including skeletal muscle development. MiR-214 is an miRNA that is differentially expressed in porcine embryonic muscle and adult skeletal muscle, suggesting that miR-214 may be related to embryonic myogenesis. In this study, the myoblast cell line C2C12 was used for functional analysis of miR-214 in vitro. The results showed that miR-214 was expressed both in myoblasts and in myotubes and was upregulated during differentiation. After treatment with an miR-214 inhibitor and culturing in differentiation medium, myoblast differentiation was repressed, as indicated by the significant downregulation of expression of the myogenic markers myogenin and myosin heavy chain (MyHC). Interestingly, myoblast proliferation was also repressed when cells were transfected with an miR-214 inhibitor and cultured in growth medium by real-time proliferation assay and cell cycle analysis. Our results showed that miR-214 regulates both proliferation and differentiation of myoblasts depending on the conditions.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r² = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.     

Identification and profiling of microRNAs from skeletal muscle of the common carp

The common carp is one of the most important cultivated species in the world of freshwater aquaculture. The cultivation of this species is particularly productive due to its high skeletal muscle mass; however, the molecular mechanisms of skeletal muscle development in the common carp remain unknown. It has been shown that a class of non-coding ～22 nucleotide RNAs called microRNAs (miRNAs) play important roles in vertebrate development. They regulate gene expression through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNAs and thereby cause translational repression or mRNA destabilization. Intriguingly, the role of miRNAs in the skeletal muscle development of the common carp remains unknown. In this study, a small-RNA cDNA library was constructed from the skeletal muscle of the common carp, and Solexa sequencing technology was used to perform high throughput sequencing of the library. Subsequent bioinformatics analysis identified 188 conserved miRNAs and 7 novel miRNAs in the carp skeletal muscle. The miRNA expression profiling showed that, miR-1, miR-133a-3p, and miR-206 were specifically expressed in muscle-containing organs, and that miR-1, miR-21, miR-26a, miR-27a, miR-133a-3p, miR-206, miR-214 and miR-222 were differentially expressed in the process of skeletal muscle development of the common carp. This study provides a first identification and profiling of miRNAs related to the muscle biology of the common carp. Their identification could provide clues leading towards a better understanding of the molecular mechanisms of carp skeletal muscle development.     

Sox9-Regulated miRNA-574-3p Inhibits Chondrogenic Differentiation of Mesenchymal Stem Cells

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.     

miR-143-3p促进C2C12成肌细胞分化

MicroRNAs (miRNAs) 是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用 real-time PCR 检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p 的模拟物和特异性抑制剂分别处理细胞,采用 real-time PCR 和 Western印迹分别检测成肌因子 MyoG和成肌标志基因 MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达 miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC 的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分化被显著抑制.检测miR-143-3p对MyHC各亚型表达的影响发现,miR-143-3p表达的变化并不直接影响MyHC各亚型的表达.以上结果说明, miR-143-3p在骨骼肌和成肌细胞中均有表达,能够促进C2C12成肌细胞分化,但并不直接调控MyHCs的表达.