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MicroRNAs (miRNAs) play an essential role in many biological processes. In this study, miRNAs in the skeletal muscle of normal and intrauterine growth retardation (IUGR) neonatal piglets were identified by sequencing, and canonical miRNAs were functionally validated in vitro. A total of 403 miRNAs were identified in neonatal piglet skeletal muscle, among them 30 and 46 miRNAs were upregulated and downregulated in IUGR pigs, respectively. Upregulated miRNAs were mainly enriched in propanoate metabolism, endocytosis, beta-Alanine metabolism, gap junction, and tumor necrosis factor signaling pathway. Down-regulated miRNAs were mainly enriched in chemical carcinogenesis—receptor activation, endocytosis, MAPK signaling pathway, insulin resistance, and EGFR tyrosine kinase inhibitor resistance. Co-expression network analysis of umbilical cord blood and skeletal muscle miRNAs showed that the miR-29 family is an essential regulator of IUGR pigs. The dual-luciferase reporter system showed that IGF1 and CCND1 were target genes of the miR-29 family. Transfection of IUGR pig umbilical cord blood exosomes and miR-29a mimic significantly inhibited cell proliferation and promoted the expression of cellular protein degradation marker genes Fbxo32 and Trim63. In summary, these results enrich the regulatory network of miRNAs involved in skeletal muscle development in IUGR animals.

Mammalian adipose tissue can be divided into white and brown adipose tissue based on its colour, location, and cellular structure. Certain conditions, such as sympathetic nerve excitement, can induce the white adipose adipocytes into a new type of adipocytes, known as beige adipocytes. The process, leading to the conversion of white adipocytes into beige adipocytes, is called white fat browning. The dynamic balance between white and beige adipocytes is closely related to the body’s metabolic homeostasis. Studying the signal transduction pathways of the white fat browning might provide novel ideas for the treatment of obesity and alleviation of obesity-related glucose and lipid metabolism disorders. This article aimed to provide an overview of recent advances in understanding white fat browning and the role of BAT in lipid metabolism.

BackgroundGene transcripts or mRNAs and long noncoding RNAs (lncRNAs) are differentially expressed during porcine skeletal muscle development. However, only a few studies have been conducted on skeletal muscle transcriptome in pigs based on timepoints according to the growth curve for porcine. Here, we investigated gene expression in Qingyu pigs at three different growth stages: of the inflection point with the maximum growth rate (MGI), inflection point of gradual increase stage to rapid increasing stage (GRI) and inflection point of rapid increasing stage to slowly increasing stage (RSI). Subsequently, we explored gene expression profiles during muscle development at the MGI, GRI and RSI stages by Ribo-Zero RNA sequencing. ResultsQingyu pigs reached the MGI, GRI and RSI stages at 156.40, 23.82 and 288.97 days of age with 51.73, 3.14 and 107.03 kg body weight, respectively. A total of 14,530 mRNAs and 11,970 lncRNAs were identified at the three stages, and 645, 323 differentially expressed genes (DEGs) and 696, 760 differentially expressed lncRNAs (DELs) were identified in the GRI vs. MGI, RSI vs. MGI comparisons. Functional enrichment analysis revealed that genes involved in immune system development and energy metabolism (mainly relate to amino acid, carbohydrate and lipid) were enriched at the GRI and MGI stages, respectively, whereas genes involved in energy and lipid metabolism were enriched at the RSI stage. We further characterized G1430, an abundant lncRNA. The full-length sequence (316 nt) of lncRNA G1430 was determined by rapid amplification of cDNA ends (RACE). Subcellular distribution analysis by quantitative real-time PCR (qRT-PCR) revealed that G1430 is a cytoplasmic lncRNA. Binding site prediction and dual luciferase assay showed that lncRNA G1430 directly binds to microRNA 133a (miR-133a). Conclusion These findings indicate lncRNAs and a certain lncRNA G1430 involved in the regulatory mechanism during pig muscle development. Our findings provide the basis for further investigation of the regulatory mechanisms and molecular genetics of muscle development in pigs.

Cardiac hypertrophy is a common pathological condition and an independent risk factor that triggers cardiovascular morbidity. As an important epigenetic regulator, miRNA is widely involved in many biological processes. In this study, miRNAs expressed in rat hearts that underwent isoprenaline-induced cardiac hypertrophy were identified using high-throughput sequencing, and functional verification of typical miRNAs was performed using rat primary cardiomyocytes. A total of 623 miRNAs were identified, of which 33 were specifically expressed in cardiac hypertrophy rats. The enriched pathways of target genes of differentially expressed miRNAs included the FoxO signaling pathway, dopaminergic synapse, Wnt signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, and Hippo signaling pathway. Subsequently, miR-144 was the most differentially expressed miRNA and was subsequently selected for in vitro validation. Inhibition of miR-144 expression in primary myocardial cells caused up-regulation of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The dual luciferase reporter system showed that ANP may be a target gene of miR-144. Long non-coding RNA myocardial infarction associated transcript (LncMIAT) is closely related to heart disease, and here, we were the first to discover that LncMIAT may act as an miR-144 sponge in isoproterenol-induced cardiac hypertrophy. Taken together, these results enriched the understanding of miRNA in regulating cardiac hypertrophy and provided a reference for preventing and treating cardiac hypertrophy.

Obesity and diabetes mellitus have become major health problems worldwide. In recent years, genistein has been found to be capable of inhibiting obesity and alleviating insulin resistance. However, the molecular mechanism of genistein against obesity is still not fully understood. In this study, we used 3T3-L1 preadipocytes and obese mice as models to explore the molecular mechanism of genistein against obesity. We found that genistein can inhibit obesity and downregulate the expression of miR-222 in mouse adipose tissue. In 3T3-L1 preadipocytes, treatment with miR-222 inhibitor or genistein reduced the expression of miR-222 and promoted lipid decomposition, while miR-222 treatment increased the expression of miR-222 and inhibited lipolysis. Moreover, the dual-luciferase reporter assay system confirmed that BTG2 and adipor1 are the target genes of miR-222. Experiments conducted in vitro and in vivo suggest that genistein may regulate lipid metabolism in the adipose tissue of obese mice by regulating the expression of miR-222 and its target genes, BTG2 and adipor1. Our findings provide a new epigenetic mechanism underpinning the ability of genistein to resist obesity. These results may provide a reference point for the dietary treatment of obesity and type 2 diabetes mellitus.

Skeletal muscle atrophy is a complex degenerative disease characterized by decreased skeletal muscle mass, skeletal muscle strength, and function. MicroRNAs (miRNAs) are a potential therapeutic target, and natural products that regulate miRNA expression may be a safe and effective treatment strategy for muscle atrophy. Previous studies have shown beneficial effects of genistein treatment on muscle mass and muscle atrophy, but the mechanism is not fully understood. Differential co-expression network analysis revealed that miR-222 was upregulated in multiple skeletal muscle atrophy models. Subsequent in vitro (C2C12 myoblasts) and in vivo (C57BL/6 mice) experiments showed that genistein could alleviate dexamethasone-induced muscle atrophy and downregulate the expression of miR-222 in muscle tissue and C2C12 myotubes. The dual-luciferase reporter assay system confirmed that IGF1 is a target gene of miR-222 and is regulated by genistein. In C2C12 myotubes, both dexamethasone and miR-222 overexpression promoted muscle atrophy, however, this function was significantly reduced after genistein treatment. Furthermore, we also observed that both genistein and miR-222 antagomiR could significantly inhibit dexamethasone-induced muscle atrophy in vivo. These results suggest that miR-222 may be involved in the regulation of genistein on muscle atrophy, and genistein and miR-222 may be used to improve muscle health.

Genistein is an isoflavone phytoestrogen that has been shown to improve obesity; however, the underlying molecular mechanisms involved therein have not been clearly elucidated. In this study, we administered genistein to high-fat diet-induced obese mice to investigate its effect on hepatic gluconeogenesis. The results showed that genistein treatment significantly inhibited body weight gain, hyperglycemia, and adipose and hepatic lipid deposition in high-fat diet-induced obese mice. Glucose tolerance test (GTT), insulin tolerance test (ITT) and pyruvate tolerance test (PTT) showed that genistein treatment significantly inhibited gluconeogenesis and improved insulin resistance in obese mice. In addition, this study also found that genistein could promote the expression of miR-451 in vitro and in vivo, and the dual-luciferase reporter system showed that G6pc (glucose-6-phosphatase) may be a target gene of miR-451. Both genistein treatment and in vivo injection of miR-451 agomir significantly inhibited gluconeogenesis and inhibited the expression of G6pc and Gk (glycerol kinase, a known target gene of miR-451). In conclusion, genistein may inhibit gluconeogenesis in obese mice by regulating the expression of Gk and G6pc through miR-451. These results may provide insights into the functions of miR-451 and food-derived phytoestrogens in ameliorating and preventing gluconeogenesis-related diseases.

As a type of non-coding RNA, microRNAs are widely involved in the biological processes of animals. In the present study, the expression of miR-152 in glycolytic muscle fibers (Longissimus thoracis, LT) was lower than that of oxidative muscle fibers (Psoas major, PM). Using dual luciferase assay, miR-152 was shown to target muscle pyruvate kinase (PKM) to perform biological functions. Moreover, overexpression of miR-152 in primary porcine cells inhibited PKM gene expression and reduced lactic acid production in cells, whereas inhibition of miR-152 expression promoted PKM gene expression and increased lactic acid production. Correlation analysis showed that the expression of miR-152 was significantly positively correlated with the ultimate pH of LT after slaughter, while the expression of the PKM gene was significantly negatively correlated with the final pH of LT. In vivo and in vitro experiments discussed herein suggest that miR-152 may affect muscle pH by targeting the expression of the PKM gene. Our findings enrich the understanding of the genetic regulatory network that influences pork quality.

Obesity due to excessive lipid accumulation is closely associated with metabolic diseases such as type 2 diabetes, insulin resistance and inflammation. Therefore, a detailed understanding of the molecular mechanisms that underlie adipogenesis is crucial to develop treatments for diseases related to obesity. Here, we found that the microRNA-204-5p (miR-204-5p) was expressed at low levels in fat tissues from obese mice fed long-term with a high-fat diet (HFD). Overexpression or inhibition of miR-204-5p in vitro in 3T3-L1 preadipocytes significantly inhibited or promoted 3T3-L1 proliferation, respectively, an effect mediated by regulating cell proliferation factors. miR-204-5p also induced preadipocyte apoptosis by directly targeting the 3' UTR region of Bcl-2, reducing the constitutive suppression of Bcl-2 on p53-dependent apoptosis. Interestingly, overexpression of miR-204-5p during adipocyte differentiation significantly increased the number of oil red O+ cells, triglyceride accumulation and the expression of markers associated with adipocyte differentiation. In contrast, inhibition of miR-204-5p had the opposite effect on 3T3-L1 adipocyte differentiation. Luciferase activity assays and qRT-PCR showed that miR-204-5p regulates adipocyte differentiation by negatively regulating KLF3, a negative regulator of lipogenesis. Taken together, our findings showed that miR-204-5p inhibits proliferation and induces apoptosis of preadipocytes by regulating Bcl-2, but also promotes adipocyte differentiation by targeting KLF3.

MicroRNAs (miRNAs) are small, non-coding RNAs that affect the post-transcriptional regulation of a variety of biological pathways. A previous study found out that miR-200b was important in the metabolism of obesity, but the mechanism of miR-200b regulateing adipogenesis is not fully understood.. In the present study, we observed that miR200b expression decreased both in obese mice and during the process of 3T3-L1 adipogenic differentiation. Overexpression of miR-200b promoted adipocyte proliferation by regulating the expression of cell cycle-regulated factors.Furthermore, overexpression of miR-200b reduced lipid accumulation and triglyceride content in adipocytes, and accompanied by a marked decrease in the expression of adipocyte-specific genes involved in fatty acid synthesis. Luciferase activity assays confirmed that miR-200b regulated adipocyte differentiation by directly targeting KLF4, and miR-200b suppressed the expression of KLF4 in both mRNA level and protein level. Moreover, the expression levels of downstream genes of KLF4, such as C/EBPβ, C/EBPα, KLF5 and PPARγ, were decreased in 3T3-L1 cells by miR-200b overexpression. Taken together, these results indicate that miR-200b may be involved in adipogenesis and could be a target for therapeutic intervention in obesity and metabolic syndrome.

The development of skeletal muscle is a complex process involving the proliferation, differentiation, apoptosis, and changing of muscle fiber types in myoblasts. Many reports have described the involvement of microRNAs in the myogenesis of myoblasts. In this study, we found that the expression of miR-152 was gradually down-regulated during myoblast proliferation, but gradually up-regulated during the differentiation of myoblasts. Transfection with miR-152 mimics restrained cell proliferation and decreased the expression levels of cyclin E, CDK4, and cyclin D1, but promoted myotube formation and significantly increased the mRNA expression levels of MyHC, MyoD, MRF4, and MyoG in C2C12 myoblasts. However, treatment with miR-152 inhibitors promoted cell proliferation and restrained differentiation. Moreover, over-expression of miR-152 significantly decreased E2F3 production in C2C12 myoblasts. A luciferase assay confirmed that miR-152 could bind to the 3' UTR of E2F3. In conclusion, this study showed that miR-152 inhibited proliferation and promoted myoblast differentiation by targeting E2F3.

Skeletal muscle is the most abundant and a highly plastic tissue of the mammals, especially when it comes to regenerate after trauma, but there is limited information about the mechanism of muscle repair and its regeneration. In the present study, we found that miR-204 is downregulated after skeletal muscle injury. In vitro experiments showed that over-expression of miR-204 by transfecting with miR-204 mimics suppressed C2C12 cell proliferation, migration, and blocked subsequent differentiation, whereas inhibition of miR-204 by transfecting with miR-204 inhibitor showed the converse effects. Furthermore, through the dual luciferase reporter system, we demonstrated that miR-204 can target the 3’UTR regions of Pax7, IGF1, and Mef2c and inhibit their expression. Taken together, our results suggest that Pax7, IGF1, and Mef2c are the target genes of miR-204 in the process of myoblasts proliferation, cell migration, and differentiation, respectively, and may contribute to mouse skeletal muscle regeneration. Our results may provide new ideas and references for the skeletal muscle study and may also provide therapeutic strategies of skeletal muscle injury.

Effective, targeted therapy for chronic liver disease nonalcoholic steatohepatitis (NASH) is imminent. MicroRNAs (miRNAs) are a potential therapeutic target, and natural products that regulate miRNA expression may be a safe and effective treatment strategy for liver disease. Here, we investigated the functional role of miR-451 and the therapeutic effects of genistein in the NASH mouse model. MiR-451 was downregulated in various types of liver inflammation, and subsequent experiments showed that miR-451 regulates liver inflammation via IL1&beta, Genistein is a phytoestrogen with anti-inflammatory and anti-oxidant effects. Interestingly, we found that the anti-inflammatory effects of genistein were related to miR-451 and was partially antagonized by the miR-451 inhibitor. MiR-451 overexpression or genistein treatment inhibited IL1&beta, expression and inflammation. Taken together, this study shows that miR-451 has a protective effect on hepatic inflammation, and genistein can be used as a natural promoter of miR-451 to ameliorate NASH.

Adipogenesis is a complex biological process and the main cause of obesity. Recently, microRNAs (miRNAs), a class of small endogenous non-coding RNAs, have been proven to play an important role in adipogenesis by the post-transcriptional regulation of target genes. In this current study, we observed an increment of miR-152 expression during the process of 3T3-L1 cell audiogenic differentiation. A functional analysis indicated that the overexpression of miR-152 inhibited pre-adipocyte proliferation and suppressed the expression of some cell cycle-related genes. Moreover, the overexpression of miR-152 promoted lipid accumulation in 3T3-L1 preadipocytes accompanied by increase of the expression of some pro-audiogenic genes. Additionally, a dual-luciferase reporter assay demonstrated lipoprotein lipase (LPL) was a direct target gene of miR-152 during preadipocyte differentiation. Further analysis showed that miR-152 was positively correlated with adipogenesis and intramuscular fat formation in vivo. Taken together, our findings suggest that miR-152 could suppress 3T3-L1 preadipocyte proliferation, whereas it could promote 3T3-L1 preadipocyte differentiation by negatively regulating LPL. The findings indicate that miR-152 might have a therapeutic significance for obesity and obesity-related metabolic syndrome.

The biochemical and functional differences between oxidative and glycolytic muscles could affect human muscle health and animal meat quality. However, present understanding of the epigenetic regulation with respect to lncRNAs and circRNAs is rudimentary. Here, porcine oxidative and glycolytic skeletal muscles, which were at the growth curve inflection point, were sampled to survey variant global expression of lncRNAs and circRNAs using RNA-seq. A total of 4046 lncRNAs were identified, including 911 differentially expressed lncRNAs (p &lt, 0.05). The cis-regulatory analysis identified target genes that were enriched for specific GO terms and pathways (p &lt, 0.05), including the oxidation-reduction process, glycolytic process, and fatty acid metabolic. All these were closely related to different phenotypes between oxidative and glycolytic muscles. Additionally, 810 circRNAs were identified, of which 137 were differentially expressed (p &lt, 0.05). Interestingly, some circRNA-miRNA-mRNA networks were found, which were closely linked to muscle fiber-type switching and mitochondria biogenesis in muscles. Furthermore, 44.69%, 39.19%, and 54.01% of differentially expressed mRNAs, lncRNAs, and circRNAs respectively were significantly enriched in pig quantitative trait loci (QTL) regions for growth and meat quality traits. This study reveals a mass of candidate lncRNAs and circRNAs involved in muscle physiological functions, which may improve understanding of muscle metabolism and development from an epigenetic perspective.

In skeletal muscle, the composition of the fiber types has a profound impact on athletic performance, such as endurance or strength output. The proportions of muscle fiber types have also been associated with certain diseases, including dyskinesia, obesity and insulin resistance. Genistein, a natural estrogen, has been demonstrated to regulate fatty acid oxidation and insulin sensitivity in skeletal muscle. However, it is unknown whether genistein can regulate skeletal muscle fiber types. Furthermore, the mechanism of its effect on skeletal muscle energy metabolism is not entirely clear. In this study, in vivo and in vitro experiments were used to explore the effect of genistein on the muscle fiber-type transitions and muscle metabolism. The results indicated that genistein not only promotes skeletal muscle development but increases the expression of slow muscle fibers in mice as well. It was also demonstrated that genistein altered the ratios of fiber type and promoted mitochondrial biogenesis in C2C12 myoblasts. Interestingly, the expression of miR-222 was decreased by genistein, and it was demonstrated that this microRNA targets the PGC1α gene. In C2C12 myoblasts, miR-222 appears to regulate fiber type conversion and mitochondrial biogenesis. However, this function was significantly reduced following genistein treatment. These results suggest that miR-222 may be involved in the regulation of genistein on skeletal muscle fiber and muscle metabolism, and genistein may be used to improve muscle health.

Cardiomyocyte hypertrophy, a prevalent clinical condition is deeply associated with many physiological factors. The underlying mechanisms of cardiomyocyte hypertrophy are not yet fully understood. In this study, H9C2 cells were treated with genistein, miR-451 mimic, miR-451 inhibitor and isoproterenol for 24 h, to study the effect of genistein on isoproterenol-induced myocardial hypertrophy in vitro. Simultaneously, ICR mice were treated with genistein for 21 days to evaluate the effects of the phytochemical on isoproterenol-induced myocardial hypertrophy in vivo. Results showed that isoproterenol induced cardiac hypertrophy and down-regulated the expression of miR-451 and up-regulated miR-451′s target gene TIMP2. Genistein increased the expression of miR-451 and inhibited the isoproterenol-induced cardiac hypertrophy. This study explored the function of genistein from the epigenetic level, suggesting that miR-451 may play a significant role in the genistein-assisted amelioration of isoproterenol-induced cardiac hypertrophy both in vitro and in vivo.

MicroRNAs (miRNAs) are small, non-coding RNAs that have been proposed to control or fine-tune complex genetic pathways by post-transcriptional regulation of target genes. It was proved that numerous miRNAs have influence on the biology of adipocytes as well as on the function of adipose tissues. This study shows that miR-10b-5p expression was decreased in mice, rats, and human under obesity. In addition, the obtained results indicated that the expression level of miR-10b-5p was increased in 3T3-L1 pre-adipocytes without manifesting a significant role in 3T3-L1 cells proliferation. On the other hand, the downregulation of miR-10b-5p by the inhibitor played a role in 3T3-L1 cells differentiation and adipogenesis. Our results strongly suggest that Apol6 was the target gene of miR-10b-5p. The inhibition of miR-10b-5p promoted the differentiation of 3T3-L1 cells and adipogenesis by upregulating the Apol6 expression. Then, the upregulated Apol6 acted as an oncogene in certain obesity-related cancers. These results indicate that miR-10b-5p may have a therapeutic significance for obesity and obesity-related cancers.

tRNA-derived fragments (tRFs) are a new class of non-coding RNA that play an important role in regulating cellular RNA processing and protein translation. However, there is currently no study reporting the influence of tRFs on myocardial hypertrophy. In this study, we used an isoproterenol (ISO)-induced myocardial hypertrophy rat model. Small RNA (

Intramuscular fat (IMF) content and composition are considered crucial indicators of porcine meat quality. However, the molecular mechanism of porcine IMF development is still mostly unclear. Recently, new evidence suggested that microRNA (miRNAs) play important roles in porcine intramuscular adipogenesis. Previously, microRNA-125a-5p (miR-125a-5p) was identified as an important regulator of adipogenesis. In the present study, we found that the expression of miR-125a-5p is dynamically regulated during porcine intramuscular preadipocytes differentiation and that its expression levels in different porcine muscle tissues were negatively involved with IMF content. To investigate the potential function role of miR-125a-5p in IMF development, porcine intramuscular preadipocytes were collected and transfected with miR-125a-5p mimics, inhibitors, or a negative control (NC), respectively. The results showed that overexpression of miR-125a-5p promoted proliferation and inhibited differentiation of porcine intramuscular preadipocytes while inhibition of miR-125a-5p had the opposite effects. Furthermore, a luciferase reporter assay demonstrated that porcine kruppel like factor 3 (KLF13) is a target gene of miR-125a-5p during porcine intramuscular preadipocytes differentiation. Interestingly, porcine ELOVL fatty acid elongase 6 (ELOVL6), a regulator of fatty acid composition, was also identified as a target gene of miR-125a-5p during porcine intramuscular adipogenesis. Further studies show that miR-125a-5p overexpression reduced total saturated fatty acids (SFA) content and monounsaturated fatty acids (MUFA)/SFA ratios while having no significant impact on polyunsaturated fatty acids (PUFA)/SFA and n-6/n-3 ratios. Taken together, our results identified that miR-125a-5p may be a novel regulator of porcine intramuscular adipogenesis and the fatty acid composition of porcine IMF.

tRNA-derived fragments (tRFs), a novel type of non-coding RNA derived from tRNAs, play an important part in governing gene expressions at a post-transcriptional level. To date, the regulatory mechanism of tRFs governing fat deposition and adipogenesis is completely unknown. In this study, high fat diet was employed to induce an obese rat model, and tRFs transcriptome sequencing was conducted to identify differentially expressed tRFs that response to obesity. We found out that tRFGluTTC, which promoted preadipocyte proliferation by increasing expressions of cell cycle regulatory factors, had the highest fold change in the 296 differentially expressed tRFs. Moreover, tRFGluTTC also suppressed preadipocyte differentiation by reducing triglyceride content and lipid accumulation, and by decreasing expressions of genes that related to fatty acid synthesis. According to results of luciferase activity analysis, tRFGluTTC directly targeted Kruppel-like factor (KLF) 9, KLF11, and KLF12, thus significantly suppressing mRNA expressions of these target genes. Moreover, tRFGluTTC suppressed adipogenesis, accompanying by suppressing expressions of adipogenic transcription factors (aP2, PPARγ, and C/EBPα). In conclusion, these results imply that tRFGluTTC may act as a novel epigenetic molecule regulating adipogenesis and could provide a new strategy for the intervention treatment of obesity.