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Emerging studies have indicated that long non-coding RNAs (lncRNAs) play important roles in skeletal muscle growth and development. Nevertheless, it remains challenging to understand the function and regulatory mechanisms of these lncRNAs in muscle biology and associated diseases. Here, we identify a novel lncRNA, Mir22hg, that is significantly upregulated during myoblast differentiation and is highly expressed in skeletal muscle. We validated that Mir22hg promotes myoblast differentiation in vitro. Mechanistically, Mir22hg gives rise to mature microRNA (miR)-22-3p, which inhibits its target gene, histone deacetylase 4 (HDAC4), thereby increasing the downstream myocyte enhancer factor 2C (MEF2C) and ultimately promoting myoblast differentiation. Furthermore, in vivo, we documented that Mir22hg knockdown delays repair and regeneration following skeletal muscle injury and further causes a significant decrease in weight following repair of an injured tibialis anterior muscle. Additionally, Mir22hg gives rise to miR-22-3p to restrict HDAC4 expression, thereby promoting the differentiation and regeneration of skeletal muscle. Given the conservation of Mir22hg between mice and humans, Mir22hg might constitute a promising new therapeutic target for skeletal muscle injury, skeletal muscle atrophy, as well as other skeletal muscle diseases.

The alteration in skeletal muscle fiber is a critical factor affecting livestock meat quality traits and human metabolic diseases. Long non-coding RNAs (lncRNAs) are a diverse class of non-coding RNAs with a length of more than 200 nucleotides. However, the mechanisms underlying the regulation of lncRNAs in skeletal muscle fibers remain elusive. To understand the genetic basis of lncRNA-regulated skeletal muscle fiber development, we performed a transcriptome analysis to identify the key lncRNAs affecting skeletal muscle fiber and meat quality traits on a pig model. We generated the lncRNA expression profiles of fast-twitch Biceps femoris (Bf) and slow-twitch Soleus (Sol) muscles and identified the differentially expressed (DE) lncRNAs using RNA-seq and performed bioinformatics analyses. This allowed us to identify 4581 lncRNA genes among six RNA libraries and 92 DE lncRNAs between Bf and Sol which are the key candidates for the conversion of skeletal muscle fiber types. Moreover, we detected the expression patterns of lncRNA MSTRG.42019 in different tissues and skeletal muscles of various development stages. In addition, we performed a correlation analyses between the expression of DE lncRNA MSTRG.42019 and meat quality traits. Notably, we found that DE lncRNA MSTRG.42019 was highly expressed in skeletal muscle and its expression was significantly higher in Sol than in Bf, with a positive correlation with the expression of Myosin heavy chain 7 (MYH7) (r = 0.6597, p = 0.0016) and a negative correlation with meat quality traits glycolytic potential (r = &minus, 0.5447, p = 0.0130), as well as drip loss (r = &minus, 0.5085, p = 0.0221). Moreover, we constructed the lncRNA MSTRG.42019&ndash, mRNAs regulatory network for a better understanding of a possible mechanism regulating skeletal muscle fiber formation. Our data provide the groundwork for studying the lncRNA regulatory mechanisms of skeletal muscle fiber conversion, and given the importance of skeletal muscle fiber types in muscle-related diseases, our data may provide insight into the treatment of muscular diseases in humans.

The meiosis-specific recombinase Dmc1 is a member of the Rad51 family that is required for the pairing of homologous chromosomes, formation of crossovers and recombination during meiosis. However, ...

Summary Inhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium., Graphical Abstract, Highlights • Activin A and BMP4 expand potency of mouse ESCs • ASCs are hypermethylated and with stable genomic imprints • ASCs developmentally closed to E4.5–E6.5 in vivo epiblast • Hypermethylated ASCs directly derived from blastocyst by ABC/L medium, In this article, Bao and colleagues show that ASCs cultured in Activin A and BMP4, and in the absence of MEK1/2 inhibitor, possess reversible epigenetic changes that extend their developmental potency, distinct transcriptome pattern, and a stable hypermethylated epigenome. Importantly, they also derived novel hypermethylated ASCs from blastocysts in ABC/L medium.

Circular RNA (circRNA) is endogenous non-coding RNA (ncRNA) with a covalently closed circular structure. It is mainly generated through RNA alternative splicing or back-splicing. CircRNA is known in the majority of eukaryotes and very stable. However, knowledge of the circRNA involved in regulating cashmere fineness is limited. Skin samples were collected from Liaoning cashmere goats (LCG) and Inner Mongolia cashmere goats (MCG) during the anagen period. For differentially expressed circRNAs, RNA sequencing was performed, and the analysis led to an identification of 17 up-regulated circRNAs and 15 down-regulated circRNAs in LCG compared with MCG skin samples. In order to find the differentially expressed circRNAs in LCG, we carried out qPCRs on 10 candidate circRNAs in coarse type skin of LCG (CT-LCG) and fine type skin of LCG (FT-LCG). Four circRNAs: ciRNA128, circRNA6854, circRNA4154 and circRNA3620 were confirmed to be significantly differential expression in LCG. Also, a regulatory network of circRNAs-miRNAs was bioinformatically deduced and may help to understand molecular mechanisms of potential circRNA involvement in regulating cashmere fineness.

Objective As one of the most important metabolic organs, the liver plays vital roles in modulating the lipid metabolism. This study was to compare miRNA expression profiles of the Large White liver between two different developmental periods and to identify candidate miRNAs for lipid metabolism. Methods Eight liver samples were collected from White Large of 70-day fetus (P70) and of 70-day piglets (D70) (with 4 biological repeats at each development period) to construct sRNA libraries. Then the eight prepared sRNA libraries were sequenced using Illumina next-generation sequencing technology on HiSeq 2500 platform. Results As a result, we obtained 346 known and 187 novel miRNAs. Compared with the D70, 55 down- and 61 up-regulated miRNAs were shown to be significantly differentially expressed (DE). Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis indicated that these DE miRNAs were mainly involved in growth, development and diverse metabolic processes. They were predicted to regulate lipid metabolism through adipocytokine signaling pathway, mitogen-activated protein kinase, AMP-activated protein kinase, cyclic adenosine monophosphate, phosphatidylinositol 3 kinase/protein kinase B, and Notch signaling pathway. The four most abundantly expressed miRNAs were miR-122, miR-26a and miR-30a-5p (miR-122 only in P70), which play important roles in lipid metabolism. Integration analysis (details of mRNAs sequencing data were shown in another unpublished paper) revealed that many target genes of the DE miRNAs (miR-181b, miR-145-5p, miR-199a-5p, and miR-98) might be critical regulators in lipid metabolic process, including acyl-CoA synthetase long chain family member 4, ATP-binding casette A4, and stearyl-CoA desaturase. Thus, these miRNAs were the promising candidates for lipid metabolism. Conclusion Our study provides the main differences in the Large White at miRNA level between two different developmental stages. It supplies a valuable database for the further function and mechanism elucidation of miRNAs in porcine liver development and lipid metabolism.

The current study examined the liver transcriptomic profiles of the Large White different in developmental periods. It was performed on pigs of two developmental stages: 70-day fetus (P70) and 70-day piglets (D70). The objective of the study was to identify genes associated with Large White liver lipid metabolism, growth and development. We sequenced eight sRNA libraries of liver samples from four Large White at P70 and D70 respectively. We totally obtained 19,202 genes. 4916 of them were found to be differentially expressed (DEGs) (p 0.05, fold change ≥ 1), of which 2502 were up-regulated and 2414 were down-regulated. GO enrichment and KEGG pathway analysis indicated that ACACA, ACADM, ACAA2 and HADH were simultaneously enriched in diverse pathways related to lipid metabolism, and so they were considered to be the promising candidate genes which could affect the porcine liver lipid metabolism. Notably, the gene insulin-like growth factor 1 (IGF1) which participated in somatotropic axis signaling was found to be up-regulated in D70 compared with P70. miRWalk and TargetScan softwares were used to screen the miRNAs which bound to the 3' untranslated region (3'UTR) of IGF1. After integration analysis with miRNAs sequencing data, miR-18b and miR-130b-3p were selected for further study. MiR-18b and miR-130b-3p were down-regulated in D70 compared with P70. Dual luciferase assays indicated that miR-18b and miR-130b-3p could obviously decrease (p 0.05) the fluorescence activity of the group transfected with the wild-type vector of IGF1 3'UTR, while the relative luciferase activity of the group transfected with the mutant vector of IGF1 3'UTR did not change significantly. Taken together, it indicated that miR-18b and miR-130b-3p could target IGF1 directly.

Summary Naive pluripotency can be maintained in medium with two inhibitors plus leukemia inhibitory factor (2i/LIF) supplementation, which primarily affects canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these three supplements alone is sufficient to maintain naive self-renewal remains unclear. Here we show that LIF alone in medium is sufficient for adaptation of 2i/L-ESCs to embryonic stem cells (ESCs) in a hypermethylated state (L-ESCs). Global transcriptomic analysis shows that L-ESCs are close to 2i/L-ESCs and in a stable state between naive and primed pluripotency. Notably, our results demonstrate that DNA methyltransferases (DNMTs) play an important role in LIF-dependent mouse ESC adaptation and self-renewal. LIF-dependent ESC adaptation efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast stem cells, L-ESCs contribute to somatic tissues and germ cells in chimeras. L-ESCs cultured under such simple conditions as in this study would provide a more conducive platform to clarify the molecular mechanism of ESCs in in vitro culture., Graphical Abstract, Highlights • LIF alone supports ESC self-renewal and pluripotency in chemically defined media • L-ESCs re-establish the epigenetic state in LIF adaptation • DNMTs are important for LIF adaptation and L-ESC self-renewal • L-ESCs contribute to somatic tissues and germ cells in chimeras, In this article, Bao and colleagues show that LIF alone supports ESC self-renewal and pluripotency in chemically defined media. L-ESCs exhibit distinct transcriptional features and re-establish DNA hypermethylation. Moreover, DNMTs are important for L-ESC adaptation and self-renewal. Notably, L-ESCs also contribute to somatic tissues and germ cells in chimeras.

Muscle fibers are closely related to human diseases and livestock meat quality. However, the genetics basis of microRNAs (miRNAs) in regulating muscle fibers is not completely understood. In this study, we constructed the whole genome-wide miRNA expression profiles of porcine fast-twitch muscle [biceps femoris (Bf)] and slow-twitch muscle [soleus (Sol)], and identified hundreds of miRNAs, including four skeletal muscle-highly expressed miRNAs, ssc-miR-378, ssc-let-7f, ssc-miR-26a, and ssc-miR-27b-3p. Moreover, we identified 63 differentially expressed (DE) miRNAs between biceps femoris vs. soleus, which are the key candidate miRNAs regulating the skeletal muscle fiber types. In addition, we found that the expression of DE ssc-miR-499-5p was significantly correlated to the expression of Myoglobin (r = 0.6872, P < 0.0001) and Myosin heavy chain 7 (MYH7; r = 0.5408, P = 0.0020), and pH45 min (r = 0.3806, P = 0.0380) and glucose content (r = -0.4382, P = 0.0154); while the expression of DE ssc-miR-499-3p was significantly correlated to the expression of Myoglobin (r = 0.5340, P = 0.0024) and pH45 min (r = 0.4857, P = 0.0065). Taken together, our data established a sound foundation for further studies on the regulatory mechanisms of miRNAs in skeletal muscle fiber conversion and meat quality traits in livestock, and could provide a genetic explanation of the role of miRNAs in human muscular diseases.

Intramuscular fat (IMF) content is a crucial indicator of meat quality. Circular RNAs (circRNAs) are a large class of endogenous RNAs that are involved in many physiological processes. However, the expression and function of circRNA in IMF in the donkey remains unresolved. Here we performed an expression profiling of circRNAs in the donkey longissimus dorsi muscle and identified 12,727 candidate circRNAs. Among these, 70% were derived from the exons of protein genes. Furthermore, a total of 127 differentially expressed (DE) circRNAs were identified in high (H) and low (L) IMF content groups, including 63 upregulated and 64 downregulated circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the host genes of the DE circRNAs showed that the host genes were enriched in lipid metabolism related GO terms (e.g., fatty acid beta-oxidation using acyl-CoA dehydrogenase and MLL3/4 complex), and signaling pathways (e.g., TGF-beta and lysine degradation signaling pathway). Further analyses indicated that 127 DE circRNAs were predicted to potentially interact with miRNAs, leading to the construction of circRNA-miRNA regulatory network. Multiple circRNAs can potentially function as sponges of miRNAs that regulate the differentiation of adipocytes. Our results provide valuable expression profile information for circRNA in the donkey and new insight into the regulatory mechanisms of circRNAs in the regulation of IMF content.

Glycolytic potential (GP) calculated based on glucose, glycogen, glucose-6-phosphate, and lactate contents is a critical factor for multiple meat quality characteristics. However, the genetic basis of glycolytic metabolism is still unclear. In this study, we constructed six RNA-Seq libraries using longissimus dorsi (LD) muscles from pigs divergent for GP phenotypic values and generated the whole genome-wide gene expression profiles. Furthermore, we identified 25,880 known and 220 novel genes from these skeletal muscle libraries, and 222 differentially expressed genes (DEGs) between the higher and lower GP groups. Notably, we found that the Lactate dehydrogenase B (LDHB) and Fructose-2, 6-biphosphatase 3 (PFKFB3) expression levels were higher in the higher GP group than the lower GP group, and positively correlated with GP and lactic acid (LA), and reversely correlated with pH value at 45 min postmortem (pH45min). Besides, LDHB and PFKFB3 expression were positively correlated with drip loss measured at 48 h postmortem (DL48h) and drip loss measured at 24 h postmortem (DL24h). Collectively, we identified a serial of DEGs as the potential key candidate genes affecting GP and found that LDHB and PFKFB3 are closely related to GP and GP-related traits. Our results lay a solid basis for in-depth studies of the regulatory mechanisms on GP and GP-related traits in pigs.

Naïve pluripotency can be maintained by the 2i/LIF supplements (CHIR99021, PD0325901 and LIF), which primarily affect canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these tripartite supplements alone is sufficient to maintain naïve self-renewal remain unclear. Here we show that LIF alone is sufficient to induce reprogramming of 2i/LIF cultured ESCs (2i/L-ESCs) to ESCs with hypermethylated state (L-ESCs). In vitro, upon withdrawal of 2i, 2i/L-ESCs overcome the epigenetic barrier and DNA hypermethylated, which accompanies transcriptional changes and subsequent establishment of epigenetic memory. Global transcriptome features also show that L-ESCs are close to 2i/L-ESCs and in a stable state between naïve and primed pluripotency. Notably, our results demonstrate that DNA methylation was indispensable for LIF-dependent mouse ESCs reprogramming and self-renew. LIF-dependent ESCs reprogramming efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast and EpiSCs, L-ESCs contribute to somatic tissues and germ cells in chimaeras. Such simple culture system of ESCs is more conducive to clarify the molecular mechanism of ESCs in vitro culture.SignificanceEmbryonic stem cell (ESCs) exhibit naïve pluripotency which reflects their ability to contribute to all embryonic lineages upon injection into blastocyst. ESCs were originally derived by co-culture with feeder cells and fetal calf serum. In this manuscript, we took a detailed approach to dissect the roles of LIF alone in ESC reprogramming of 2i/LIF cultured ESCs (2i/L-ESCs). Here, for the first time, we derived stable hypermethylated pluripotent ESCs under culture of LIF alone (L-ESCs). We further assessed L-ESCs properties both in vitro and in vivo, and provide molecular insights to the mechanism which allows LIF alone to maintain pluripotency and a hypermethylated state. We believe these findings are novel and valuable for future ESCs study.

The intronic microRNA, miR-125b, plays a vital role in promyelocytic and hematopoietic stem cells, and in the development and apoptosis of cancer cells. In this study, we showed that miR-125b regulates granulosa cell (GC) apoptosis in the yak ovary. Bioinformatic analyses and luciferase reporter assays demonstrated that bone morphogenetic protein receptor type 1B (BMPR1B) is an miR-125b target. miR-125b overexpression induced apoptosis in yak GC, and affected the mRNA and protein expression of BMPR1B and the ratio of Bcl2/Bax. Silencing of miR-125b decreased the rate of yak GC apoptosis and increased the ratio of Bcl2/Bax. In addition, the effects of an miR-125b inhibitor were overturned by cotransfection with siRNA-BMPR1B2 (siRNA-299) in yak GC. Together, these results demonstrated that miR-125b regulates GC apoptosis in the yak ovary by targeting BMPR1B.

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.

Small molecules discovered during the recent years can be used to regulate the growth of embryonic stem cells (ES cells). Chicken blastodermal cells (cBCs) play an important role in both basic and transgenic researches as an important ES cell. However, the regulatory mechanism of small molecules involved in the self-renewal and pluripotency of cBCs remains unknown. This study revealed that the small molecule, SC1, can maintain cBCs in an undifferentiated, pluripotent state in serum- and feeder-free E8 media without leukaemia inhibitory factor. Furthermore, SC1 inhibits downregulation of pluripotency-related genes caused by retinoic acid and promotes the proliferation of cBCs. Furthermore, the results of this study indicated that SC1 functions by inhibiting ERK1 phosphorylation and promoting Akt phosphorylation, thus promoting the expression of pluripotency-related genes and maintaining the pluripotency of cBCs. The results also demonstrated that SC1 sustains the self-renewal capacity and pluripotency of cBCs cells by inhibiting ERK1 phosphorylation and promoting Akt phosphorylation. This kind of regulatory mechanism might be conserved in avian ES cells. Other molecules, similar to SC1, might provide insights into the molecular mechanisms that control the fate of stem cells and ultimately help in-vivo stem cell biology and therapy.

The Sine oculis homeobox 1 (Six1) gene is important for skeletal muscle growth and fiber specification; therefore, it is considered as a promising candidate gene that may influence porcine growth and meat quality traits. Nevertheless, the association of Six1 with these processes and the mechanisms regulating its expression remain unclear. The objectives of this study were to identify variant sites of Six1 in different pig breeds, conduct association analysis to evaluate the relationship between polymorphisms of these variants and porcine production traits in Pietrain × Duroc × Landrace × Yorkshire commercial pigs, and explore the potential regulatory mechanisms of Six1 affecting production traits. A total of 12 variants were identified, including 10 single- nucleotide variations (SNVs), 1 insertion- deletion (Indel), and 1 (AC)n microsatellite. Association analysis demonstrated that the SNV, g.1595A>G, was significantly associated with meat color (redness, a*); individuals with the G allele had greater a* values (P < 0.05). Moreover, our results demonstrated that the (AC)n polymorphism in the Six1 promoter was significantly associated with weaning weight (P < 0.05), carcass weight (P < 0.05), and thoracic and lumbar back fat (P < 0.01).In addition, we found that the (AC)n variant was closely related with Six1 expression levels and demonstrated this polymorphism on promoter activity by in vitro experiments. Overall, this study provides novel evidence for elucidating the effects of Six1 on porcine production traits as promising candidate and describes two variants with these traits, which are potential reference markers for pig molecular breeding. In addition, our data on the relationship between porcine Six1 expression and the polymorphic (AC)n microsatellite in its promoter may facilitate similar studies in other species.

Skeletal muscle is a major component of body mass and plays a central role in the control of whole-body metabolism in humans and animals. Therefore, elucidation of the underlying mechanisms of skeletal growth and development are expected to lead to the discovery of novel genes and pathways related to muscle disease. miR-206, a skeletal muscle-specific microRNA, plays a crucial role in myogenesis; however, miR-206 is known to function in myogenic differentiation, whether or not it affects muscle cells' proliferation, and the underlying mechanisms are unknown. In this study, we investigated the effect of miR-206 on muscle cell proliferation and differentiation, as well as its effect on myofiber type conversion using mouse C2C12 myoblasts. The results showed that overexpression of miR-206 inhibited cell proliferation and promoted muscle cell differentiation, but it did not affect myofiber type conversion. Intriguingly, we found that overexpression of miR-206 suppressed muscle cell proliferation and induced cell cycle arrest in G

Prox1 is involved in muscle fiber conversion, adult-onset obesity, and type 2 diabetes. However, information regarding porcine Prox1 and its relationship with meat quality traits is still unknown. In this study, we characterized the full-length cDNA and proximal promoter of two transcript variants of porcine Prox1. Moreover, Prox1 was expressed abundantly in the skeletal muscle and its expression was higher in the soleus muscle than that in the biceps femoris muscle. Its expression pattern in the high and low meat color (redness) value a* groups was similar to that of myoglobin and MyHC I, but opposed to that of MyHC IIB. Importantly, there was a significant positive correlation between Prox1 expression and meat color (redness) value a* (r = 0.3845, p = 0.0394), and a significant negative correlation between Prox1 expression and drip loss (r = &minus, 0.4204, p = 0.0232), as well as the ratio of MyHC IIB to MyHC I expression (r = &minus, 0.3871, p = 0.0380). In addition, we found that the polymorphisms of three closely linked SNPs in Prox1 promoter 1 were significantly associated with pH24h in a pig population. Taken together, our data provide valuable insights into the characteristics of porcine Prox1 and indicate that Prox1 is a promising candidate gene affecting meat quality traits.

Accumulating evidence indicates a critical role for T cells and relevant cytokines in the pathogenesis of systemic lupus erythematosus (SLE). However, the specific contribution of T cells together with the related circulating cytokines in disease pathogenesis and organ involvement is still not clear. In the current study, we investigated relevant molecule expressions and cytokine levels in blood samples from 49 SLE patients and 22 healthy control subjects. The expression of HLA-DR and costimulatory molecules on T cells was evaluated by flow cytometry. Concentrations of serum C-reactive protein, erythrocyte sedimentation rate, anti-double-stranded DNA (anti-dsDNA) antibody, total lgG, complement 3, and complement 4 were measured. Serum cytokines and chemokines were measured by a cytometric bead array assay. Elevated frequencies of HLA-DR+ T cells and ICOS+ T cells were observed in SLE patients with positive anti-dsDNA antibodies compared with those in healthy controls (P<0.001). The expression of HLA-DR+ T cells was positively correlated with SLEDAI (r=0.15, P<0.01). Furthermore, levels of serum IL-6, MCP-1, TNFRI, IL-10, IL-12, and CCL20 were higher in SLE patients compared with healthy controls. In addition, patients with hematologic manifestations displayed elevated frequencies of HLA-DR+ T cells and ICOS+ T cells. Patients with renal manifestations had a decreased frequency of TIGIT+ T cells. These results suggested a dysregulated T cell activity and cytokine expression profiles in SLE subjects. We also developed a chemokine and cytokine profiling strategy to predict the activity of SLE, which has clinical implication for better monitoring the flares and remission during the course of SLE and for assessing therapeutic interventions.

FK506-binding protein 6 (FKBP6) is essential for meiosis during mammalian spermatogenesis. However, the molecular regulation of FKBP6 during spermatogenesis remains unclear. In the present study, we performed molecular characterization of the meiosis-specific gene FKBP6 in yak testes. Yak FKBP6 encodes a polypeptide of 295 amino acid residues with an FK506-binding domain (FKBP_C) and three tetratricopeptide repeat domains. The methylation level of the FKBP6 promoter in testes was significantly higher in cattle-yak with male sterility than in yak, and the FKBP6 promoter was methylated in liver tissues in which FKBP6 is not expressed. FKBP6 promoter activity was significantly decreased after treatment with the M.SssI methyltransferase in vitro. Furthermore, the FKBP6 gene was remarkably activated in bovine mammary epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine. Taken together, our results demonstrate for the first time that the FKBP6 promoter is differentially methylated in testes; together with the functional promoter analysis, this suggests that methylation of this promoter may contribute to cattle-yak male infertility.

MicroRNAs (miRNAs) are endogenous short non-coding RNAs and exert their function by targeting mRNAs of genes. miRNA-206 (miR-206) is exclusively expressed in adult skeletal muscles and plays an important role in myogenesis. However, the regulatory mechanisms of miR-206 in myoblasts proliferation and differentiation are still limited. In this study, we validated that Notch3 is a direct target gene of mouse miR-206 using dual-luciferase reporter assay and miR-206-overexpressed experiments. Furthermore, we demonstrated that the negative effect of overexpression of miR-206 and knockdown of Notch3 on C2C12 cells proliferation, and found that the effect was produced by controlling the transition of G0/G1 and S phase in C2C12 cell cycle. In summary, our results provide direct evidences that miR-206 regulates skeletal muscle cells proliferation and cell cycle arrest partly by targeting the Notch3 gene, which strengthens our understanding of regulatory mechanisms on miR-206 in skeletal muscle growth and development.

In livestock, glycolytic potential (GP) is a critical indicator for evaluating the meat quality. To date, two major genes protein kinase AMP-activated γ3 non-catalytic subunit gene (PRKAG3) and phosphorylase kinase catalytic subunit gamma 1(PHKG1), and corresponding cause mutations influencing GP have been confirmed in pigs. Therefore, the aim of this study to identify the novel candidate genes and variations related to GP-related traits using a four-hybrid pig model [Pietrain (P)× Duroc (D)] ×[(Landrace) ×(Yorkshire)]. We totally constructed six RNA-seq libraries using longissimus dorsi (LD) muscles, and each library contained two higher GP (H) or two lower GP (L) individuals. A total of 525, 698 and 135 differentially expressed genes (DEGs) were identified between H11 vs L11, H9 vs L9, and H5 vs L5 groups using PossionDis method, respectively. Notably, we found 97 non-redundant DEGs were mapped to GP related QTLs from three paired comparison groups. Moreover, 69 DEGs were identified between H (H11, H9 and H5) and L (L11, L9 and L5) groups using NOIseq method. Additionally, 1,076 potential specific SNPs were figured out between H and L groups, and approximately 40 large Indels with a length ≥ 5bp were identified in each sequencing library. In conclusion, our data provide foundation for further confirming the key genes and the functional mutations affecting GP-related traits in pigs, and also pave the way for elucidating the underling molecular regulatory mechanisms of glycogen metabolism in future study. Moreover, this study might provide valuable information for study on human glycogen storage diseases.

Intramuscular fat (IMF) content is an important indicator for meat quality evaluation. However, the key genes and molecular regulatory mechanisms affecting IMF deposition remain unclear. In the present study, we identified 75 differentially expressed genes (DEGs) between the higher (H) and lower (L) IMF content of pigs using transcriptome analysis, of which 27 were upregulated and 48 were downregulated. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEG perilipin-1 (PLIN1) was significantly enriched in the fat metabolism-related peroxisome proliferator-activated receptor (PPAR) signaling pathway. Furthermore, we determined the expression patterns and functional role of porcine PLIN1. Our results indicate that PLIN1 was highly expressed in porcine adipose tissue, and its expression level was significantly higher in the H IMF content group when compared with the L IMF content group, and expression was increased during adipocyte differentiation. Additionally, our results confirm that PLIN1 knockdown decreases the triglyceride (TG) level and lipid droplet (LD) size in porcine adipocytes. Overall, our data identify novel candidate genes affecting IMF content and provide new insight into PLIN1 in porcine IMF deposition and adipocyte differentiation.

Follicular atresia mainly results from apoptosis of granulosa cells (GCs). Our previous microRNA array data indicated that the miRNA let-7g level increases significantly during porcine ovary follicular atresia. It is uncertain if GCs apoptosis is mediated by microRNA let-7g. In this study, the expression levels of the apoptosis-associated genes CASP3, BAX and BIM were significantly upregulated when let-7g mimic was transfected into porcine GCs, and the anti-apoptotic genes BCL-2 and MCL-1 were significantly downregulated. The apoptosis rate was measured by flow cytometry, and our results indicated that let-7g significantly enhanced GCs apoptosis. In further studies, we found that overexpression of let-7g induced the expression of FoxO1 in GCs and led to nuclear accumulation of dephosphorylated FoxO1. In addition, the effect of let-7g on FoxO1 expression and dephosphorylation resulted from repression of the expression of the MAP3K1 gene in porcine GCs. The site on MAP3K1 mRNA targeted by let-7g was confirmed by luciferase reporter assay. The anti-apoptotic effect of MAP3K1 was validated by silencing MAP3K1 using small interfering RNA technology. In conclusion, our data indicate that let-7g induces porcine GCs apoptosis by inhibiting the MAP3K1 gene, which promotes FoxO1 expression and dephosphorylation with nuclear accumulation.

Mei1 is required for the homologous recombination of meiosis during the mammalian spermatogenesis. However, the knowledge about bovine Mei1 (bMei1) is still limited. In the present study, we cloned and characterized the bMei1, and investigated the epigenetic regulatory mechanism of bMei1 expression in vivo and in vitro. The full length coding region of bMei1 was 3819bp, which encoded a polypeptide of 1272 amino acids. Real-time PCR showed that the mRNA expression level of bMei1 in the testis of cattle-yak with meiotic arrest and male infertility was significantly decreased as compared with cattle (P

The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

Meat color is considered to be the most important indicator of meat quality, however, the molecular mechanisms underlying traits related to meat color remain mostly unknown. In this study, to elucidate the molecular basis of meat color, we constructed six cDNA libraries from biceps femoris (Bf) and soleus (Sol), which exhibit obvious differences in meat color, and analyzed the whole-transcriptome differences between Bf (white muscle) and Sol (red muscle) using high-throughput sequencing technology. Using DEseq2 method, we identified 138 differentially expressed genes (DEGs) between Bf and Sol. Using DEGseq method, we identified 770, 810, and 476 DEGs in comparisons between Bf and Sol in three separate animals. Of these DEGs, 52 were overlapping DEGs. Using these data, we determined the enriched GO terms, metabolic pathways and candidate genes associated with meat color traits. Additionally, we mapped 114 non-redundant DEGs to the meat color QTLs via a comparative analysis with the porcine quantitative trait loci (QTL) database. Overall, our data serve as a valuable resource for identifying genes whose functions are critical for meat color traits and can accelerate studies of the molecular mechanisms of meat color formation.

Drip loss, one of the most important meat quality traits, is characterized by low heritability. To date, the genetic factors affecting the drip loss trait have not been clearly elucidated. The objective of this study was to identify critical candidate genes affecting drip loss. First, we generated a Pietrain × Duroc × Landrace × Yorkshire commercial pig population and obtained phenotypic values for the drip loss trait. Furthermore, we constructed two RNA libraries from pooled samples of longissimus dorsi muscles with the highest (H group) and lowest (L group) drip loss and identified the differentially expressed genes (DEGs) between these extreme phenotypes using RNA-seq technology. In total, 25 883 genes were detected in the H and L group libraries, and none was specifically expressed in only one library. Comparative analysis of gene expression levels found that 150 genes were differentially expressed, of which 127 were upregulated and 23 were downregulated in the H group relative to the L group. In addition, 68 drip loss quantitative trait loci (QTL) overlapping with 63 DEGs were identified, and these QTL were distributed mainly on chromosomes 1, 2, 5 and 6. Interestingly, the triadin (TRDN) gene, which is involved in muscle contraction and fat deposition, and the myostatin (MSTN) gene, which has a role in muscle growth, were localized to more than two drip loss QTL, suggesting that both are critical candidate genes responsible for drip loss.

Non-primate mammals have two deleted azoospermia (DAZ) family genes, DAZL and Boule; genes in this family encode RNA-binding proteins essential for male fertility in diverse animals. Testicular DAZL transcription is regulated by epigenetic factors such as DNA methylation. However, nothing is known about the epigenetic regulation of Boule. Here, we explored the role of DNA methylation in the regulation of the bovine Boule (bBoule) gene. We found that a long CpG island (CGI) in the bBoule promoter was hypermethylated in the testes of cattle-yak hybrids with low bBoule expression, whereas cattle had relatively low methylation levels (P < 0.01), and there was no difference in the methylation level in the short CGI of the gene body between cattle and cattle-yak hybrids (P > 0.05). We identified a 107 bp proximal core promoter region of bBoule. Intriguingly, the differences in the methylation level between cattle and cattle-yak hybrids were larger in the core promoter than outside the core promoter. An in vitro methylation assay showed that the core promoter activity of bBoule decreased significantly after M.SssI methylase treatment (P < 0.01). We also observed dramatically increased bBoule transcription in bovine mammary epithelial cells (BMECs) after treatment with the methyltransferase inhibitor 5-Aza-dC. Taken together, our results establish that methylation status of the core promoter might be involved in testicular bBoule transcription, and may provide new insight into the epigenetic regulation of DAZ family genes and clinical insights regarding male infertility.

Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced granulosa cell (GC) apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. In this study, we found expression of the p53-upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 subfamily protein, in ovarian GCs during oxidative stress. By overexpression and knockdown of Forkhead box O1 (FoxO1), we found that FoxO1 regulates PUMA at the protein level. Moreover, as c-Jun N-terminal kinase (JNK) has been shown to activate FoxO1 by promoting its nuclear import, we used a JNK inhibitor to reduce FoxO1 activation and detected decreased PUMA messenger RNA expression and protein levels during oxidative stress. In addition, in vivo oxidative stress-induced upregulation of PUMA was found following injection of 3 nitropropionic acid in mice. In conclusion, oxidative stress increases PUMA expression regulated by FoxO1 in follicular GCs.

Follicle-stimulating hormone (FSH) plays a crucial role in animal reproduction and exerts its physiological functions by interacting with the FSH receptor (FSHR). The FSHR is exclusively expressed in granulose cells in the ovary and its expression level is closely related to granulose cell differentiation and follicle maturation. In mammal, most of the follicles undergo atresia, while follicle atresia is mainly caused by granulosa cell apoptosis. However, knowledge on the transcriptional regulatory mechanisms of the porcine FSHR gene in granulosa cell is still limited. In this study, approximately 2.1kb of the proximal promoter sequence of the porcine FSHR gene were obtained by genome walking, and the regulatory elements and transcription factors in the porcine FSHR promoter sequence were predicted. Furthermore, the core promoter region (−1195/−598) of the porcine FSHR gene was identified using a luciferase assay. Subsequently, the relationship between expression levels of the porcine FSHR gene and histone H3K9 acetylation levels around the core promoter region (−787/−572) in vivo and in vitro were analyzed. Our results showed that an increased FSHR gene expression level was accompanied with an increase in histone H3K9 acetylation levels, suggesting that histone H3K9 acetylation could regulate the expression of the porcine FSHR gene.

Boule is responsible for meiotic arrest of sperms and male sterility during mammalian spermatogenesis. In the present study, we first identified yak b-Boule gene and its two alternative splice variants. The full length coding region of yak b-Boule is 888bp and encodes a 295-amino acid protein with a typical RNA-recognition motif (RRM) and a Deleted in Azoospermia (DAZ) repetitive sequence motif. Two alternative splice variants of yak b-Boule were generated following the consensus "GT-AG" rule and named b-Boule1 (36bp deletion in exon 3) and b-Boule2 (deletion of integral exon 7), respectively. In male yak, b-Boule, b-Boule1 and b-Boule2 were found to be exclusively expressed in the testes at a ratio of 81:0.1:1. Intriguingly, the mRNA expression levels of b-Boule and b-Boule1 in yak testis were significantly higher than those in cattle-yak, although no significant difference was observed for b-Boule2 expression between the yak and cattle-yak. These results suggest that b-Boule gene, which is partially regulated by alternative splicing, may be involved in the process of yak spermatogenesis.

The sine oculis homeobox 1 (Six1) gene encodes an evolutionarily conserved transcription factor. In the past two decades, much research has indicated that Six1 is a powerful regulator participating in skeletal muscle development. In this review, we summarized the discovery and structural characteristics of Six1 gene, and discussed the functional roles and molecular mechanisms of Six1 in myogenesis and in the formation of skeletal muscle fibers. Finally, we proposed areas of future interest for understanding Six1 gene function.

In mammalian ovaries, follicular atresia occurs periodically and destroys almost all the follicles in the ovary. Follicle-stimulating hormone (FSH) acts as the primary survival factor during follicular atresia by preventing apoptosis in granulosa cells. FoxO1 is a critical factor in promoting follicular atresia and granulosa cell apoptosis. FSH inhibits the induction of FoxO1. In this report, we investigated the role of FSH-FoxO1 pathway in mouse follicular atresia. FSH dampened stress-induced apoptosis and the expression of FoxO1 and pro-apoptosis genes in mouse granulosa cells (MGCs). In contrast, overexpression of FoxO1 inhibited the viability of MGCs and induced the expression of endogenous FoxO1. The signaling cascades involved in regulating FoxO1 activity upon FSH treatment were identified using FSH signaling antagonists. Blocking protein kinase A (PKA), phosphatidylinositol-3 kinase (PI3K) or protein kinase B (AKT) restored the upregulation of FoxO1 and apoptotic signals, which was suppressed by FSH. Moreover, inhibition of PKA or PI3K impaired FSH-induced AKT activity, but inactivation of PI3K or AKT had little effect on PKA activity in the presence of FSH. Correspondingly, constitutive activation of FoxO1 (all three AKT sites were replaced by alanines) also promoted MGC apoptosis despite FSH administration. Furthermore, both luciferase reporter assays and chromatin immunoprecipitation assays showed that FoxO1 directly bound to a FoxO-recognized element site within the FoxO1 promoter and contributed to the regulation of FoxO1 expression in response to FSH. Taken together, we propose a novel model in which FSH downregulates FoxO1-dependent apoptosis in MGCs by coordinating the PKA–PI3K–AKT–FoxO1 axis and FoxO1–FoxO1 positive feedback.