Comprehensive variation discovery and recovery of missing sequence in the pig genome using multiple de novo assemblies.

Uncovering genetic variation through resequencing is limited by the fact that only sequences with similarity to the reference genome are examined. Reference genomes are often incomplete and cannot represent the full range of genetic diversity as a result of geographical divergence and independent demographic events. To more comprehensively characterize genetic variation of pigs (Sus scrofa), we generated de novo assemblies of nine geographically and phenotypically representative pigs from Eurasia. By comparing them to the reference pig assembly, we uncovered a substantial number of novel SNPs and structural variants, as well as 137.02-Mb sequences harboring 1737 protein-coding genes that were absent in the reference assembly, revealing variants left by selection. Our results illustrate the power of whole-genome de novo sequencing relative to resequencing and provide valuable genetic resources that enable effective use of pigs in both agricultural production and biomedical research.

miR-27a-5p Attenuates Hypoxia-induced Rat Cardiomyocyte Injury by Inhibiting Atg7.

Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality worldwide. AMI triggers a hypoxic microenvironment and induces extensive myocardial injury, including autophagy and apoptosis. MiRNAs, which are a class of posttranscriptional regulators, have been shown to be involved in the development of ischemic heart diseases. We have previously reported that hypoxia significantly alters the miRNA transcriptome in rat cardiomyoblast cells (H9c2), including miR-27a-5p. In the present study, we further investigated the potential function of miR-27a-5p in the cardiomyocyte response to hypoxia, and showed that miR-27a-5p expression was downregulated in the H9c2 cells at different hypoxia-exposed timepoints and the myocardium of a rat AMI model. Follow-up experiments revealed that miR-27a-5p attenuated hypoxia-induced cardiomyocyte injury by regulating autophagy and apoptosis via Atg7, which partly elucidated the anti-hypoxic injury effects of miR-27a-5p. Taken together, this study shows that miR-27a-5p has a cardioprotective effect on hypoxia-induced H9c2 cell injury, suggesting it may be a novel target for the treatment of hypoxia-related heart diseases.

Comprehensive Analysis of lncRNAs and circRNAs Reveals the Metabolic Specialization in Oxidative and Glycolytic Skeletal Muscles.

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 < 0.05). The cis-regulatory analysis identified target genes that were enriched for specific GO terms and pathways (p < 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 < 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.

Spontaneous single nucleotide polymorphism in porcine microRNA-378 seed region leads to functional alteration.

Sequence variation in a microRNA (miRNA) seed region can influence its biogenesis and effects on target mRNAs; however, in mammals, few seed region mutations leading to functional alterations have been reported to date. Here, we report the identification of a single nucleotide polymorphism (SNP) with functional consequence located in the seed region of porcine miR-378. In vitro analysis of this rs331295049 A17G SNP showed significantly up-regulated expression of the mature miR-378 (miR-378/G). In silico target prediction indicated that the SNP would modulate secondary structure and result in functional loss affecting >85% of the known target genes of the wild-type miR-378 (miR-378/A), and functional gain affecting >700 new target genes, and dual-luciferase reporter assay verified this result. This report of a SNP in the seed region of miR-378 leads to functional alteration and indicates the potential for substantive functional consequences to the molecular physiology of a mammalian organism.

Transcriptome-wide N 6 -methyladenosine methylome profiling of porcine muscle and adipose tissues reveals a potential mechanism for transcriptional regulation and differential methylation pattern.

BACKGROUND: N 6 -methyladenosine (m6A) is the most prevalent internal form of modification in messenger RNA in higher eukaryotes and potential regulatory functions of reversible m6A methylation on mRNA have been revealed by mapping of m6A methylomes in several species. m6A modification in active gene regulation manifests itself as altered methylation profiles in a tissue-specific manner or in response to changing cellular or species living environment. However, up to date, there has no data on m6A porcine transcriptome-wide map and its potential biological roles in adipose deposition and muscle growth. METHODS: In this work, we used methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) technique to acquire the first ever m6A porcine transcriptome-wide map. Transcriptomes of muscle and adipose tissues from three different pig breeds, the wild boar, Landrace, and Rongchang pig, were used to generate these maps. RESULTS: Our findings show that there were 5,872 and 2,826 m6A peaks respectively, in the porcine muscle and adipose tissue transcriptomes. Stop codons, 3'-untranslated regions, and coding regions were found to be mainly enriched for m6A peaks. Gene ontology analysis revealed that common m6A peaks in nuclear genes are associated with transcriptional factors, suggestive of a relationship between m6A mRNA methylation and nuclear genome transcription. Some genes showed tissue- and breed-differential methylation, and have novel biological functions. We also found a relationship between the m6A methylation extent and the transcript level, suggesting a regulatory role for m6A in gene expression. CONCLUSION: This comprehensive map provides a solid basis for the determination of potential functional roles for RNA m6A modification in adipose deposition and muscle growth.

Coat colour phenotype of Qingyu pig is associated with polymorphisms of melanocortin receptor 1 gene.

OBJECTIVE: Qingyu pig, a Chinese indigenous pig breed, exhibits two types of coat colour phenotypes, including pure black and white with black spotting respectively. Melanocortin receptor 1 (MC1R) and agouti signaling protein (ASIP) are two widely reported pivotal genes that significantly affect the regulation of coat colour. The objectives of this study were to investigate whether the polymorphisms of these two genes are associated with coat colour and analyze the molecular mechanism of the coat colour separation in Qingyu pig. METHODS: We studied the phenotype segregation and used polymerase chain reaction amplification and Sanger sequencing to investigate the polymorphism of MC1R and ASIP in 121 Qingyu pigs, consisting of 115 black and 6 white with black spotted pigs. RESULTS: Coat colour of Qingyu pig is associated with the polymorphisms of MC1R but not ASIP. We only found 2 haplotypes, EQY and Eqy , based on the 13 observed mutations from MC1R gene. Among which, Eqy presented a recessive inheritance mode in black spotted Qingyu pigs. Further analysis revealed a g.462-463CC insertion that caused a frameshift mutation and a premature stop codon, thus changed the first transmembrane domain completely and lost the remaining six transmembrane domains. Altogether, our results strongly support that the variety of Qingyu pig's coat colour is related to MC1R. CONCLUSION: Our findings indicated that black coat colour in Qingyu pig was dominant to white with black spotted phenotype and MC1R gene polymorphism was associated with coat colour separation in Qingyu pig.

Effect of miR-143-3p on C2C12 myoblast differentiation.

MicroRNAs are a class of 18-22 nucleotide non-coding RNAs that modulate gene expression by associating with the 3' untranslated regions of mRNAs. A large number of microRNAs are involved in the regulation of myoblast differentiation, many of which remain undiscovered. In this study, we found that miR-143-3p was upregulated during C2C12 myoblast differentiation and over-expression of miR-143-3p significantly inhibited the relative expression levels of MyoD, MyoG, myf5, and MyHC genes, especially in the later stages of differentiation. In addition, miR-143-3p inhibited expression of genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and ß-catenin. These results indicate that miR-143-3p represents a new myogenic differentiation-associated microRNA that can inhibit C2C12 myoblast differentiation, especially in the later stages of differentiation.

Dynamic changes in genes related to glucose uptake and utilization during pig skeletal and cardiac muscle development.

Skeletal and cardiac muscle have important roles in glucose uptake and utilization. However, changes in expression of protein coding genes and miRNAs that participate in glucose metabolism during development are not fully understood. In this study, we investigated the expression of genes related to glucose metabolism during muscle development. We found an age-dependent increase in gene expression in cardiac muscle, with enrichment in heart development- and energy-related metabolic processes. A subset of genes that were up-regulated until 30 or 180 days postnatally, and then down-regulated in psoas major muscle was significantly enriched in mitochondrial oxidative-related processes, while genes that up-regulated in longissimus doris muscle was significantly enriched in glycolysis-related processes. Meanwhile, expression of energy-related microRNAs decreased with increasing age. In addition, we investigated the correlation between microRNAs and mRNAs in three muscle types across different stages of development and found many potential microRNA-mRNA pairs involved in regulating glucose metabolism.

Breed, sex and anatomical location-specific gene expression profiling of the porcine skeletal muscles.

BACKGROUND: Skeletal muscle is one of the most important economic traits in agricultural animals, especially in pigs. In the modern pig industry, lean type pigs have undergone strong artificial selection for muscle growth, which has led to remarkable phenotypic variations compared with fatty type pigs, making these different breeds an ideal model for comparative studies. RESULTS: Here, we present comprehensive gene expression profiling for the white (longissimus dorsi muscle) and the red (psoas major muscle) skeletal muscles among male and female fatty Rongchang, feral Tibetan and lean Landrace pigs, using a microarray approach. We identified differentially expressed genes that may be associated the phenotypic differences of porcine muscles among the breeds, between the sexes and the anatomical locations. We also used a clustering method to identify sets of functionally coexpressed genes that are linked to different muscle phenotypes. We showed that, compared with the white muscles, which primarily modulate metabolic processes, the red muscles show a tendency to be a risk factor for inflammation and immune-related disorders. CONCLUSIONS: This analysis presents breed-, sex- and anatomical location-specific gene expression profiles and further identified genes that may be associated with the phenotypic differences in porcine muscles among breeds, between the sexes and the anatomical locations.

Gene expression profiles of specific chicken skeletal muscles.

The chicken provides large amounts of protein for the human diet and is also used as a model organism for biomedical research. Increasing meat production is an important goal in the poultry industry and skeletal muscles have highly diverse origins, shapes, metabolic features, and physical functions. Previous gene expression atlases have largely ignored the differences among diverse types of skeletal muscles; therefore, comprehensive transcriptional maps of all skeletal muscles are needed to improve meat production traits. In this study, we sequenced 58 samples from 10 different skeletal muscles of 42-day-old White Plymouth Rock chickens. We also measured myofiber diameter and generated myofiber-type datasets of these 10 tissues. We generated 418.4 Gb high-quality bulk RNA-Seq data from four or six biological replicates of each skeletal muscle (four replicates from extraocular samples) (approximately 7.4 Gb per sample). This dataset provides valuable information for understanding the muscle fiber characteristics of White Plymouth Rock chickens. Furthermore, our data can be used as a model for heterogeneity analysis between tissues with similar properties.

MicroRNAs miR-27a and miR-143 regulate porcine adipocyte lipid metabolism.

MicroRNAs (miRNAs) are non-coding small RNAs that play roles in regulating gene expression. Some miRNAs have been classed as epigenetic regulators of metabolism and energy homeostasis. Previous reports indicated that the miRNAs miR-27a and miR-143 were involved in lipid metabolism in human and rodents. To determine the roles of porcine miR-27a and miR-143 in adipocyte lipid metabolism, porcine adipocytes were cultured and allowed to induce differentiation for 10 days. The lipid-filled adipocytes were then transfected with miRNA mimics and inhibitors. We measured how the indicators of adipogenesis and adipolysis in porcine adipocytes were affected by the over-expression and by the inhibition of both miR-27a and miR-143. The results indicated that the over-expression of miR-27a could accelerate adipolysis releasing significantly more glycerol and free fatty acids than the negative control (P < 0.001), while the mimic of miR-143 expression, promoted adipogenesis by accumulating more triglycerides (P < 0.001) in the adipocytes. In addition, we demonstrated that there was good correlation (r > 0.98, P < 0.001) between the indicators of adipolysis in cell lysates and in the culture medium.

Identification and expression pattern analysis of miRNAs in pectoral muscle during pigeon (Columba livia) development.

MicroRNAs (miRNAs) are a group of crucial regulators in the process of animal growth and development. However, little is known about the expression and function of miRNAs in pigeon muscles. To identify the miRNAs participating in the rapid development of pigeon pectoral muscles and quantitate their expression levels of pectoral muscles in different age stages, we performed miRNA transcriptome analysis in pigeon pectoral muscles by sequencing small RNAs over three different age stages (1-day old, 28 days old, and 2 years old). Dual-luciferase reporter assay was applied to validate the interaction between miRNA and its target gene. We identified 304 known miRNAs, 201 conserved miRNAs, and 86 novel miRNAs in pigeon pectoral muscles. 189 differentially expressed (DE) miRNAs were screened out during pigeon development. A short time-series expression miner (STEM) analysis indicated 89 DE miRNAs were significantly clustered in a progressively decreasing expression profile, and mainly enriched in biosynthesis-related GO categories and signaling pathways for MAPK and TGF-ß. Dual-luciferase reporter assay indicated that a progressively down-regulated miRNA (miR-20b-5p) could directly target Krüppel-like factor 3 (KLF3) gene. To sum-up, our data expand the repertoire of pigeon miRNAs and enhance understanding of the mechanisms underlying rapid development in squabs.

Dietary betaine prevents obesity through gut microbiota-drived microRNA-378a family.

Betaine is a natural compound present in commonly consumed foods and may have a potential role in the regulation of glucose and lipids metabolism. However, the underlying molecular mechanism of its action remains largely unknown. Here, we show that supplementation with betaine contributes to improved high-fat diet (HFD)-induced gut microbiota dysbiosis and increases anti-obesity strains such as Akkermansia muciniphila, Lactobacillus, and Bifidobacterium. In mice lacking gut microbiota, the functional role of betaine in preventing HFD-induced obesity, metabolic syndrome, and inactivation of brown adipose tissues are significantly reduced. Akkermansia muciniphila is an important regulator of betaine in improving microbiome ecology and increasing strains that produce short-chain fatty acids (SCFAs). Increasing two main members of SCFAs including acetate and butyrate can significantly regulate the levels of DNA methylation at host miR-378a promoter, thus preventing the development of obesity and glucose intolerance. However, these beneficial effects are partially abolished by Yin yang (YY1), a common target gene of the miR-378a family. Taken together, our findings demonstrate that betaine can improve obesity and associated MS via the gut microbiota-derived miR-378a/YY1 regulatory axis, and reveal a novel mechanism by which gut microbiota improve host health.

Comprehensive Analysis of mRNA and lncRNA Transcriptomes Reveals the Differentially Hypoxic Response of Preadipocytes During Adipogenesis.

Local hypoxia has recently been reported to occur in the white adipose tissue (WAT) microenvironment during obesity. Adipocytes have a unique life cycle that reflects the different stages of adipogenesis in the WAT niche. Long non-coding RNAs (lncRNAs) play an important role in the cellular response to hypoxia. However, the differentially hypoxic responses of preadipocytes during adipogenesis and the potential role of lncRNAs in this process remain to be elucidated. Here, we evaluated the differentially hypoxic responses of primary hamster preadipocytes during adipogenesis and analyzed mRNA and lncRNA expression in same Ribo-Zero RNA-seq libraries. Hypoxia induced HIF-1α protein during adipogenesis and caused divergent changes of cell phenotypes. A total of 10,318 mRNAs were identified to be expressed in twenty libraries (five timepoints), and 3,198 differentially expressed mRNAs (DE mRNAs) were detected at five timepoints (hypoxia vs. normoxia). Functional enrichment analysis revealed the shared and specific hypoxia response pathways in the different stages of adipogenesis. Hypoxia differentially modulated the expression profile of adipose-associated genes, including adipokines, lipogenesis, lipolysis, hyperplasia, hypertrophy, inflammatory, and extracellular matrix. We also identified 4,296 lncRNAs that were expressed substantially and detected 1,431 DE lncRNAs at five timepoints. Two, 3, 5, 13, and 50 DE mRNAs at D0, D1, D3, D7, and D11, respectively, were highly correlated and locus-nearby DE lncRNAs and mainly involved in the cell cycle, vesicle-mediated transport, and mitochondrion organization. We identified 28 one-to-one lncRNA-mRNA pairs that might be closely related to adipocyte functions, such as ENSCGRT00015041780-Hilpda, TU2105-Cdsn, and TU17588-Ltbp3. These lncRNAs may represent the crucial regulation axis in the cellular response to hypoxia during adipogenesis. This study dissected the effects of hypoxia in the cell during adipogenesis, uncovered novel regulators potentially associated with WAT function, and may provide a new viewpoint for interpretation and treatment of obesity.

Genistein reverses isoproterenol-induced cardiac hypertrophy by regulating miR-451/TIMP2.

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.

Expression profiling analysis for genes related to meat quality and carcass traits during postnatal development of backfat in two pig breeds.

The competitive equilibrium of fatty acid biosynthesis and oxidation in vivo determines porcine subcutaneous fat thickness (SFT) and intramuscular fat (IMF) content. Obese and lean-type pig breeds show obvious differences in adipose deposition; however, the molecular mechanism underlying this phenotypic variation remains unclear. We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with meat quality and carcass traits in backfat at five growth stages (1-5 months) of Landrace (a leaner, Western breed) and Taihu pigs (a fatty, indigenous, Chinese breed). Variance analysis (ANOVA) revealed that differences in the expression of 25 genes in Landrace pigs were significant (FDR adjusted permutation, P<0.05) among 5 growth stages. Gene class test (GCT) indicated that a gene-group was very significant between 2 pig breeds across 5 growth stages (P (ErmineJ)<0.01), which consisted of 23 genes encoding enzymes and regulatory proteins associated with lipid and steroid metabolism. These findings suggest that the distinct differences in fat deposition ability between Landrace and Taihu pigs may closely correlate with the expression changes of these genes. Clustering analysis revealed a very high level of significance (FDR adjusted, P<0.01) for 2 gene expression patterns in Landrace pigs and a high level of significance (FDR adjusted, P<0.05) for 2 gene expression patterns in Taihu pigs. Also, expression patterns of genes were more diversified in Taihu pigs than those in Landrace pigs, which suggests that the regulatory mechanism of micro-effect polygenes in adipocytes may be more complex in Taihu pigs than in Landrace pigs. Based on a dynamic Bayesian network (DBN) model, gene regulatory networks (GRNs) were reconstructed from time-series data for each pig breed. These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of adipose metabolism between the two pig breeds; from these results, some potential key genes could be identified. Quantitative, real-time RT-PCR (QRT-PCR) was used to verify the microarray data for five modulated genes, and a good correlation between the two measures of expression was observed for both 2 pig breeds at different growth stages (R=0.874+/-0.071). These results highlight some possible candidate genes for porcine fat characteristics and provide some data on which to base further study of the molecular basis of adipose metabolism.

MicroRNA-143a-3p modulates preadipocyte proliferation and differentiation by targeting MAPK7.

Adipogenesis plays a key role in increasing fat mass, which is a main characteristic for obesity, and involves preadipocyte proliferation and differentiation. Recently, more and more evidences suggested that microRNAs (miRNAs) is an important member of the regulatory network of adipogenesis. In this study, miR-143a-3p was highly expressed in adipose tissues of obese mice, and was up-regulated at the middle and last stage of 3T3-L1 adipocyte differentiation. Using mouse 3T3-L1 cells line, which is an ideal model in vitro for the study of adipogenesis, we observed that overexpression of miR-143a-3p inhibited the preadipocyte proliferation, and enhanced the preadipocyte differentiation. In contrast, the inhibition of miR-143a-3p expression promoted the preadipocyte proliferation, and inhibited the preadipocyte differentiation. Further analysis suggested that miR-143a-3p mediating preadipocyte differentiation might be involved in fatty acid metabolism. In addition, we found that miR-143-3p and PPARγ, an activator of miR-143a-3p transcription, could regulate each other. Compared with miR-143a-3p, MAPK7 played an opposite role in the proliferation and differentiation of adipocyte. Further analysis indicated that MAPK7 is a target gene of miR-143a-3p in 3T3-L1 cells, and inhibition of MAPK7 recede the effect of miR-143a-3p on preadipocyte proliferation and differentiation. Taken together, these results indicated that as a regulator of PPARγ, miR-143a-3p play an important role in adipogenesis via regulating MAPK7 and fatty acid.

Global Long Noncoding RNA and mRNA Expression Changes between Prenatal and Neonatal Lung Tissue in Pigs.

Lung tissue plays an important role in the respiratory system of mammals after birth. Early lung development includes six key stages, of which the saccular stage spans the pre- and neonatal periods and prepares the distal lung for alveolarization and gas-exchange. However, little is known about the changes in gene expression between fetal and neonatal lungs. In this study, we performed transcriptomic analysis of messenger RNA (mRNA) and long noncoding RNA (lncRNA) expressed in the lung tissue of fetal and neonatal piglets. A total of 19,310 lncRNAs and 14,579 mRNAs were identified and substantially expressed. Furthermore, 3248 mRNAs were significantly (FDR-adjusted p value ≤ 0.05, FDR: False Discovery Rate) differentially expressed and were mainly enriched in categories related to cell proliferation, immune response, hypoxia response, and mitochondrial activation. For example, CCNA2, an important gene involved in the cell cycle and DNA replication, was upregulated in neonatal lungs. We also identified 452 significantly (FDR-adjusted p value ≤ 0.05) differentially expressed lncRNAs, which might function in cell proliferation, mitochondrial activation, and immune response, similar to the differentially expressed mRNAs. These results suggest that differentially expressed mRNAs and lncRNAs might co-regulate lung development in early postnatal pigs. Notably, the TU64359 lncRNA might promote distal lung development by up-regulating the heparin-binding epidermal growth factor-like (HB-EGF) expression. Our research provides basic lung development datasets and will accelerate clinical researches of newborn lung diseases with pig models.

miR-144-3p Promotes Adipogenesis Through Releasing C/EBPα From Klf3 and CtBP2.

MicroRNAs (miRNAs), a class of small non-coding RNAs, have been proved as novel and potent regulators of adipogenesis. A previous study has found out that miR-144-3p was a biomarker of type 2 diabetes, but the role of miR-144-3p in regulating adipogenesis was still unclear. In the present study, the expression of miR-144-3p increased in obese mice and during the 3T3-L1 differentiation process. Overexpression of miR-144-3p suppressed the expression of cell cycle regulatory factors and inhibited pre-adipocytes proliferation. Besides, overexpression of miR-144-3p accelerated lipid accumulation in adipocytes and positively regulated adipogenesis, which was also accompanied by increasing the expression of genes related to fatty acid synthesis and decreasing the expression of genes involved in fatty acid oxidation. Furthermore, luciferase activity assays indicated that miR-144-3p directly targeted Klf3 and CtBP2. The process was also confirmed by the mRNA and protein expression of Klf3 and CtBP2, which were suppressed by miR-144-3p. Furthermore, miR-144-3p targeting Klf3/CtBP2 would induce C/EBPα activity by releasing corepressors (Klf3 and CtBP2) from its promoter region. Moreover, we also observed that miR-144-3p could promote adipogenesis in mice injected with miR-144-3p agomir through tail-vein injection. Taken together, these results support that miR-144-3p can facilitate adipogenesis both in vitro and in vivo, which implies that miR-144-3p could be a target for therapeutic intervention in obesity and metabolic syndrome in the future.

Long Noncoding RNA GAS5 Suppresses 3T3-L1 Cells Adipogenesis Through miR-21a-5p/PTEN Signal Pathway.

Emerging studies indicated that both long noncoding RNAs and micro-RNAs play crucial roles in the mediation of adipogenesis, which is closely linked to obesity-related diseases. However, the mechanisms of lncRNA-miRNAs coregulating in adipogenesis are still largely unknown. In this study, we determined that lncRNA growth arrest-specific 5 (GAS5) presented an opposite expression pattern with miR-21a-5p in 3T3-L1 adipocytes development. To explore the role of GAS5 in adipogenesis, pcDNA3.1-GAS5 expression vectors and GAS5-siRNAs were used to perform GAS5 overexpression and knockdown, respectively. Ectopic expression of GAS5 dramatically reduced miR-21a-5p level and suppressed the proliferation of 3T3-L1 preadipocytes, while silencing GAS5 slightly increased miR-21a-5p expression but had no significant influence on the cell viability. In addition, overexpression of GAS5 remarkably decreased the mRNA and protein levels of adipogenic marker genes, and resulted in a notable reduction of lipid accumulation. In contrast, overexpressing miR-21a-5p significantly facilitated differentiation of 3T3-L1 cells. By target gene prediction and luciferase reporter assay, we suggested that GAS5 might indirectly improve the expression of phosphatase and tensin homolog (PTEN) by repressing miR-21a-5p in a miRNA-based regulatory mechanism. Together, GAS5 plays a suppressive role in 3T3-L1 cells adipogenesis, which further highlights the importance of lncRNAs in adipogenesis.