Xin-E Shi

MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3′ untranslated regions (3′ UTRs) of messenger RNAs (mRNAs). Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

MicroRNA-199a-5p Affects Porcine Preadipocyte Proliferation and Differentiation

MicroRNAs (miRNAs), a class of small non-coding RNAs, have emerged as novel and potent regulators of adipogenesis. However, few miRNAs have been fully investigated in porcine adipogenesis, given the fact that pig is not only an apropos model of human obesity research, but also a staple meat source of human diet. In this study, we showed that miRNA-199a-5p is highly expressed in porcine subcutaneous fat deposits compared to several other tissue types and organs measured alongside. Overexpression of miR-199a-5p in porcine preadipocytes significantly promoted cell proliferation while attenuating the lipid deposition in porcine adipocytes. By target gene prediction and experimental validation, we demonstrated that caveolin-1 (Cav-1) may be a bona fide target of miR-199a-5p in porcine adipocytes, accounting for some of miR-199a-5p’s functions. Taken together, our data established a role of miR-199a-5p in porcine preadipocyte proliferation and differentiation, which is at least partially played by downregulating Cav-1.

BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI) is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

Retinol binding protein 4 affects the adipogenesis of porcine preadipocytes through insulin signaling pathways

Retinol binding protein 4 (RBP4), a novel cytokine, is mainly secreted by hepatocytes and adipocytes. RBP4 reportedly induces insulin resistance and RBP4 secretion is increased in the adipocytes of animals or humans with type 2 diabetes, obesity, and metabolic syndrome, but its role in preadipocyte differentiation remains unclear. In this study, we investigated the effect of RBP4 on the differentiation of porcine preadipocytes into adipocytes. The results suggest that RBP4 significantly suppresses the differentiation of porcine preadipocytes into adipocytes, including those treated with the hormone cocktail methylisobutylxanthine-dexamethasone-insulin. RBP4 also weakened the activity of normal threonine 308, the phosphorylation of serine/threonine kinase AKT, and downstream insulin signaling, including the mammalian target of rapamycin (mTOR) and β-catenin. Moreover, the activation of insulin signaling mediated by knockdown RBP4 in porcine preadipocytes was recovered in the suppression of LY294002. RBP4 also had a suppressive effect on the differentiation of porcine preadipocytes by decreasing the activation of insulin signaling pathways.

FGF13 regulates proliferation and differentiation of skeletal muscle by down‐regulating Spry1

Generally, the secretory forms of FGF are known to regulate cell proliferation, differentiation and morphogenesis by binding to the extracellular domain of cell surface receptors. Intracellular FGFs (FGF11‐14) are expressed principally in the nervous system. FGF13 is a microtubule‐stabilizing protein that regulates neuronal polarization and migration. Previous studies have reported high expression of FGF13 in cultures of single muscle fibres. However, functions of FGF13 in muscle development have not been explored.

An Additive Effect of Promoting Thermogenic Gene Expression in Mice Adipose-Derived Stromal Vascular Cells by Combination of Rosiglitazone and CL316,243

It is well-documented that CL316,243 (a β3 agonist) or rosiglitazone (a PPARγ agonist) can induce white adipocyte populations to brown-like adipocytes, thus increasing energy consumption and combating obesity. However, whether there is a combined effect remains unknown. In the present study, stromal vascular cells of inguinal white adipose tissue (iWAT-SVCs for short) from mice were cultured and induced into browning by CL316,243, rosiglitazone, or both. Results showed that a combination of CL316,243 and rosiglitazone significantly upregulated the expression of the core thermogenic gene Ucp1 as well as genes related with mitochondrial function (Cidea, Cox5b, Cox7a1, Cox8b, and Cycs), compared with the treatment of CL316,243 or rosiglitazone alone. Moreover, co-treatment with rosiglitazone could reverse the downregulation of Adiponectin resulting from CL316,243 stimuli alone. Taken together, a combination of rosiglitazone and CL316,243 can produce an additive effect of promoting thermogenic gene expression and an improvement of insulin sensitivity in mouse iWAT-SVCs.

Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells.

Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition.

miR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes

MicroRNAs (miRNAs) are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old) Rongchang piglets rather than their adult counterparts (180-day-old). This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3′-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes.

Differentiation of 3T3-L1 preadipocytes is inhibited under a modified ceiling culture †

Ceiling culture is an inverted and closed cell culture system which represents a novel method for exploring adipocyte characteristics and function. Although the role of ceiling culture in mature adipocyte dedifferentiation has been extensively studied, its potential effects on preadipocyte differentiation remain unclear. In this study, we established a simplified dish ceiling culture method for 3T3‐L1 preadipocytes and showed that our novel ceiling culture method could reproduce the function of the traditional flask ceiling culture. Then, we investigated the effects of ceiling culture on 3T3‐L1 preadipocyte differentiation by Oil red O staining and RT‐qPCR. The results showed that ceiling culture significantly impaired triglyceride accumulation and adipogenic marker genes expression in 3T3‐L1 preadipocytes. These findings suggest that ceiling culture inhibited 3T3‐L1 preadipocyte differentiation while inducing mature adipocytes dedifferentiation. Taken together, our data facilitate the understanding of the property of ceiling culture and promote the study of revealing the underlying mechanisms of mature adipocytes dedifferenatiation.

miR-423-5p inhibits myoblast proliferation and differentiation by targeting Sufu: GE et al.

Myoblast proliferation and terminal differentiation are the key steps of myogenesis. MicroRNAs are a class of small noncoding RNAs that play important roles in gene expression regulation. They negatively regulate gene expression by causing messenger RNA translational repression or target messenger RNA degradation. Here, we found that microRNA‐423‐5p (miR‐423‐5p) is highly expressed in both slow and fast muscles. Our gain‐of‐function study indicated that miR‐423‐5p actually plays a negative role in regulating myoblast proliferation and differentiation. We also found that miR‐423‐5p is able to inhibit the expression of suppressor of fused homolog to inactivate the expression of the marker genes in myoblast proliferation and differentiation. Taken together, our findings indicated miR‐423‐5p as a potential inhibitor of myogenesis by targeting suppressor of fused homolog in myoblast, and it also contributes to a better understanding of the microRNAs‐target gene regulatory network in different types of porcine muscle types and may benefit the practice of improving the meat quality in animal husbandry.