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.
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.
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.
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