Background Long non-coding RNAs (lncRNAs) play critical regulatory roles in diverse biological processes and diseases. While a large number of lncRNAs have been identified in skeletal muscles until now, their function and underlying mechanisms in skeletal myogenesis remain largely unclear. Methods We characterized a novel functional lncRNA designated lncMGPF (lncRNA muscle growth promoting factor) using RACE, Northern blot, fluorescence in situ hybridization and quantitative real-time PCR. Its function was determined by gene overexpression, interference, and knockout experiments in C2C12 myoblasts, myogenic progenitor cells, and an animal model. The molecular mechanism by which lncMGPF regulates muscle differentiation was mainly examined by cotransfection experiments, luciferase reporter assay, RNA immunoprecipitation, RNA pull-down, and RNA stability analyses. Results We report that lncMGPF, which is highly expressed in muscles and positively regulated by myoblast determination factor (MyoD), promotes myogenic differentiation of muscle cells in vivo and in vitro. lncMGPF knockout in mice substantially decreases growth rate, reduces muscle mass, and impairs muscle regeneration. Overexpression of lncMGPF in muscles can rescue the muscle phenotype of knockout mice and promote muscle growth of wild-type mice. Mechanistically, lncMGPF promotes muscle differentiation by acting as a molecular sponge of miR-135a-5p and thus increasing the expression of myocyte enhancer factor 2C (MEF2C), as well as by enhancing human antigen R-mediated messenger RNA stabilization of myogenic regulatory genes such as MyoD and myogenin (MyoG). We confirm that pig lncRNA AK394747 and human lncRNA MT510647 are homologous to mouse lncMGPF, with conserved function and mechanism during myogenesis. Conclusions Our data reveal that lncMGPF is a novel positive regulator of myogenic differentiation, muscle growth and regeneration in mice, pigs, and humans.

MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein a (Cebpa), peroxisome proliferatoractivated receptor g (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/b-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of b-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/b-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/b-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue.

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Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca 2+ in PPARγ pigs. Raising of Ca 2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.

Bovine astrovirus (BoAstV) belongs to genus Mamastravirus (MAstV). It can be detected in the faeces of both diarrhoeal and healthy calves. However, its prevalence, genetic diversity, and association with cattle diarrhoea are poorly understood. In this study, faecal samples of 87 diarrhoeal and 77 asymptomatic calves from 20 farms in 12 provinces were collected, and BoAstV was detected with reverse transcription-polymerase chain reaction (RT-PCR). The overall prevalence rate of this virus in diarrhoeal and asymptomatic calves was 55.17 % (95 % CI: 44.13, 65.85 %) and 36.36 % (95 % CI: 25.70, 48.12 %), respectively, indicating a correlation between BoAstV infection and calf diarrhoea (OR=2.15, P=0.024). BoAstV existed mainly in the form of co-infection (85.53 %) with one to five of nine viruses, and there was a strong positive correlation between BoAstV co-infection and calf diarrhoea (OR=2.83, P=0.004). Binary logistic regression analysis confirmed this correlation between BoAstV co-infection and calf diarrhoea (OR=2.41, P=0.038). The co-infection of BoAstV and bovine rotavirus (BRV) with or without other viruses accounted for 70.77 % of all the co-infection cases. The diarrhoea risk for the calves co-infected with BoAstV and BRV was 8.14-fold higher than that for the calves co-infected with BoAstV and other viruses (OR=8.14, P=0.001). Further, the co-infection of BoAstV/BRV/bovine kobuvirus (BKoV) might increase the risk of calf diarrhoea by 14.82-fold, compared with that of BoAstV and other viruses (OR=14.82, P <0.001). Then, nearly complete genomic sequences of nine BoAstV strains were assembled by using next-generation sequencing (NGS) method. Sequence alignment against known astrovirus (AstV) strains at the levels of both amino acids and nucleotides showed a high genetic diversity. Four genotypes were identified, including two known genotypes MAstV-28 (n=3) and MAstV-33 (n=2) and two novel genotypes designated tentatively as MAstV-34 (n=1) and MAstV-35 (n=3). In addition, seven out of nine BoAstV strains showed possible inter-genotype recombination and cross-species recombination. Therefore, our results increase the knowledge about the prevalence and the genetic evolution of BoAstV and provide evidence for the association between BoAstV infection and calf diarrhoea.

The modulation of the interaction between macrophages and Mycobacterium tuberculosis (M.tb) through microRNA during M.tb infection is increasingly capturing the attention of researchers. However, the potential role of microRNA-18b-5p (miR-18b) is not elucidated yet. In this study, miR-18b was found to be downregulated in M.tb-infected macrophage cell lines (THP-1 and RAW264.7) in time- and dose-dependent manners. Furthermore, when the miR-18b mimic and inhibitor and small interfering RNA hypoxia-inducible factor 1α (si-HIF-1α) were transfected into the macrophages separately or in combination, it was found that miR-18b targeted hypoxia-inducible factor 1α (HIF-1α). During M.tb infection, the decrease in the expression of miR-18b facilitated HIF-1α expression, which led to the increased production of pro-inflammatory cytokines, such as IL-6, resulting in decreased bacterial survival in the host cells. Moreover, the phosphorylation of p38 MAPK and NF-κB p65 was activated by the miR-18b inhibitor. Our findings expand the current understanding of the M.tb–cell interaction mechanism and provide a potential target to control M.tb infection.

Indirubin is a Chinese medicine extracted from indigo and known to be effective for treating chronic myelogenous leukemia, neoplasia, and inflammatory disease. This study evaluated the in vivo anti-inflammatory activity of indirubin in a lipopolysaccharide- (LPS-) induced mouse mastitis model. The indirubin mechanism and targets were evaluated in vitro in mouse mammary epithelial cells. In the mouse model, indirubin significantly attenuated the severity of inflammatory lesions, edema, inflammatory hyperemia, milk stasis and local tissue necrosis, and neutrophil infiltration. Indirubin significantly decreased myeloperoxidase activity and downregulated the production of tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 caused by LPS. In vitro, indirubin inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner. It also downregulated LPS-induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein and inhibitor of kappa B. In addition to its effect on the NF-κB signaling pathway, indirubin suppressed the mitogen-activated protein kinase (MAPK) signaling by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), P38, and c-jun NH2-terminal kinase (JNK). Indirubin improved LPS-induced mouse mastitis by suppressing TLR4 and downstream NF-κB and MAPK pathway inflammatory signals and might be a potential treatment of mastitis and other inflammatory diseases.

Mycobacterium tuberculosis (M. tb) can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of M. tb-infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during M. tb infection. This study aimed to investigate the roles of miR-378d in M. tb infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, M. tb infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, M. tb survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1β, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during M. tb infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with M. tb infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during M. tb infection of macrophages, miR-378d was down-regulated and functioned on decreasing M. tb intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1β, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against M. tb infection.