MicroRNA-542-3p targets Pten to inhibit the myoblasts proliferation but suppresses myogenic differentiation independent of targeted Pten.

BACKGROUND: Non-coding RNA is a key epigenetic regulation factor during skeletal muscle development and postnatal growth, and miR-542-3p was reported to be conserved and highly expressed in the skeletal muscle among different species. However, its exact functions in the proliferation of muscle stem cells and myogenesis remain to be determined. METHODS: Transfection of proliferative and differentiated C2C12 cells used miR-542-3p mimic and inhibitor. RT-qPCR, EdU staining, immunofluorescence staining, cell counting kit 8 (CCK-8), and Western blot were used to evaluate the proliferation and myogenic differentiation caused by miR-542-3p. The dual luciferase reporter analysis and rescued experiment of the target gene were used to reveal the molecular mechanism. RESULTS: The data shows overexpression of miR-542-3p downregulation of mRNA and protein levels of proliferation marker genes, reduction of EdU+ cells, and cellular vitality. Additionally, knocking it down promoted the aforementioned phenotypes. For differentiation, the miR-542-3p gain-of-function reduced both mRNA and protein levels of myogenic genes, including MYOG, MYOD1, et al. Furthermore, immunofluorescence staining immunized by MYHC antibody showed that the myotube number, fluorescence intensity, differentiation index, and myotube fusion index all decreased in the miR-542-3p mimic group, compared with the control group. Conversely, these phenotypes exhibited an increased trend in the miR-542-3p inhibitor group. Mechanistically, phosphatase and tensin homolog (Pten) was identified as the bona fide target gene of miR-542-3p by dual luciferase reporter gene assay, si-Pten combined with miR-542-3p inhibitor treatments totally rescued the promotion of proliferation by loss-function of miR-542-3p. CONCLUSIONS: This study indicates that miR-542-3p inhibits the proliferation and differentiation of myoblast and Pten is a dependent target gene of miR-542-3p in myoblast proliferation, but not in differentiation.

Integrated analysis of differently expressed microRNAs and mRNAs at different postnatal stages reveals intramuscular fat deposition regulation in goats (Capra hircus).

Intramuscular fat refers to the adipose tissue distributed in the muscle. It is an important indicator that affects the quality of goat meat, and can directly affect the tenderness and flavor of goat meat. Our previous study revealed the mRNA that may be crucial for intramuscular fat deposition during goat growth; however, how the microRNAs (miRNAs) are involved in the process is largely unclear. In the present study, a total of 401 known miRNAs and 120 goat novel miRNAs, including 110 differentially expressed (DE) miRNAs, were identified among longissimus dorsi from three growth stages (2, 9, and 24 months) by miRNA sequencing. Combining analysis of the DE mRNAs and DE miRNAs was then performed by miRDB and miRwalk, and miR-145-5p and FOXO1, miR-487b-3p, and PPARG coactivator 1 α (PPARGC1A), miR-345-3p, and solute carrier family 2 member 4 (SLC2A4), etc. were shown to closely associate with lipid metabolism, which was then validated by a correlation analysis. The final DE mRNAs were significantly enriched in fatty acid transmembrane transport, fatty acid homeostasis, apelin signaling pathway, glucagon signaling pathway, insulin signaling pathway, and AMPK signaling pathway by gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Besides, miR-145-5p showed a certain effect on goat intramuscular fat metabolism by acting on the possible target gene Forkhead Box O1 (FOXO1). These data provide some theoretical support for improving the quality of goat meat.

Neuron-derived neurotrophic factor promotes the differentiation of intramuscular and subcutaneous adipocytes in goat.

Adipocyte play an important role in human health and meat quality by influencing the tenderness, flavor, and juiciness of mutton It has been shown that neuron-derived neurotrophic factor (NENF) is closely related to energy metabolism and adipocyte differentiation in bovine. However, the role of NENF in the goats remains unclear. The aim of this study was to detect the expression of NENF in goat subcutaneous and intramuscular adipocytes, temporal expression profiles of the NENF, and overexpressed NENF on the differentiation of different adipocytes. In this study, PCR amplification successfully cloned the goat NENF gene with a fragment length of 521 bp. In addition, the time point of highest expression of NENF differed between these two adipocytes differentiation processes. Overexpression of NENF in intramuscular and subcutaneous adipocytes promoted the expression levels of differentiation markers CEBPß and SREBP, which in turn promoted the differentiation of intramuscular and subcutaneous adipocytes. This study will provide basic data for further study of the role of goats in goat adipocyte differentiation and for the final elucidation of its molecular mechanisms in regulating goat adipocyte deposition.

Effect of DHCR7 on adipocyte differentiation in goats.

Cholesterol is regarded as a signaling molecule in regulating the metabolism and function of fat cells, in which 7-Dehydrocholesterol reductase (DHCR7) is a key enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol, however, the exact function of DHCR7 in goat adipocytes remains unknown. Here, the effect of DHCR7 on the formation of subcutaneous and intramuscular fat in goats was investigated in vitro, and the result indicated that the mRNA level of DHCR7 showed a gradual downward trend in subcutaneous adipogenesis, but an opposite trend in intramuscular adipogenesis. In the process of subcutaneous preadipocytes differentiation, overexpression of DHCR7 inhibited the expression of adipocytes differentiation marker genes (CEBP/α, CEBP/ß, SREBP1 and AP2), lipid metabolism-related genes (AGPAT6, FASN, SCD1 and LPL), and the lipid accumulation. However, in intramuscular preadipocyte differentiation, DHCR7 overexpression showed a promoting effect on adipocyte differentiation marker genes (CEBP/α, CEBP/ß, PPARγ and SREBP1) and lipid metabolism-related genes (GPAM, AGPAT6, DGAT1 and SCD1) expression, and on lipid accumulation. In summary, our work demonstrated that DHCR7 played an important role in regulating adipogenic differentiation and lipid metabolism in preadipocytes in goats, which is of great significance for uncovering the underlying molecular mechanism of adipocyte differentiation and improving goat meat quality.

miR-424(322)-5p targets Ezh1 to inhibit the proliferation and differentiation of myoblasts.

The proliferation and differentiation of myoblasts are considered the key biological processes in muscle development and muscle-related diseases, in which the miRNAs involved remain incompletely understood. Previous research reported that miR-424(322)-5p is highly expressed in mouse skeletal muscle. Therefore, C2C12 cells are used as a model to clarify the effect of miR-424(322)-5p on the proliferation and differentiation of myoblasts. The data show that miR-424(322)-5p exhibits a decreasing trend upon myogenic differentiation. Overexpression of miR-424(322)-5p inhibits the proliferation of myoblasts, manifested by downregulation of proliferation marker genes ( CCNB1, CCND2, and CDK4), decreased percentage of EdU + cells, and reduced cell viability. In contrast, these phenotypes are promoted in myoblasts treated with an inhibitor of miR-424(322)-5p. Interestingly, its gain of function inhibits the expression of myogenic regulators, including MyoD, MyoG, MyHC, and Myf5. Additionally, immunofluorescence staining of MyHC and MyoD shows that overexpression of miR-424(322)-5p reduces the number of myotubes and decreases the myotube fusion index. Consistently, inhibition of its function mediated by an inhibitor promotes the expressions of myogenic markers and myotube fusion. Mechanistically, gene prediction and dual-luciferase reporter experiments confirm that enhancer of zeste homolog 1 ( Ezh1) is one of the targets of miR-424(322)-5p. Furthermore, knockdown of Ezh1 inhibits the proliferation and differentiation of myoblasts. Compared with NC and inhibitor treatment, inhibitor+si- EZH1 treatment rescues the phenotypes of proliferation and differentiation mediated by the miR-424(322)-5p inhibitor. Taken together, these data indicate that miR-424(322)-5p targets Ezh1 to negatively regulate the proliferation and differentiation of myoblasts.

Diacylglycerol acyltransferase 2 promotes the adipogenesis of intramuscular preadipocytes in goat.

Diacylglycerol acyltransferase 2 (DGAT2) is the key enzyme that catalyzes the last step of triglyceride synthesis. However, its role in intramuscular fat (IMF) deposition in goat remains unclear. The purpose of this study was to explore the role of DGAT2 in regulating goat IMF deposition. In the present study, the expression of DGAT2 was highest in goat triceps brachii, and highest on the first day after oleic acid induction in goat intramuscular preadipocytes. The overexpression of DGAT2 promoted the accumulation of lipid droplets and triglyceride synthesis, accompanied by the expression upregulation of DGAT1, TIP47, ACC and ACOX1 significantly, and expression downregulation of AGPAT6, LPIN1, LPL, HSL, ATGL and ADRP significantly. In contrast, the silencing of DGAT2 decreased the accumulation of lipid droplets, inhibited the expression of DGAT1, GPAM, ADRP, AGPAT6, LPL, HSL, ATGL, ACC, FASN, ACOX1 significantly, and enhanced that of TIP47 significantly. Overall, these data underscore DGAT2 may play a potentially important role in lipid droplets formation and triglyceride accumulation, so as to maintain intramuscular fat deposition, beyond triglyceride synthesis in goat.

PDZK1-interacting protein 1(PDZK1IP1) promotes subcutaneous preadipocyte proliferation in goats.

PDZK1-interacting protein 1(PDZK1IP1), also known as MAP17, is encoded by the PDZK1IP1 gene and is a membrane-associated protein. PDZK1IP1 have been proven to be a potent regulator of cancer cell proliferation. However, the role of PDZK1IP1 in regulating goat subcutaneous preadipocyte proliferation is unknown. Here, we cloned the full-length coding sequence of PDZK1IP1 gene, investigated the potential functional of PDZK1IP1 in goat subcutaneous preadipocyte proliferation by gaining or losing function in vitro. Our results indicated that goat PDZK1IP1 gene consists of 345 bp, encoding a protein of 114 amino acids containing a typical PDZK1IP1 (MAP17) super family domain. Overexpression of PDZK1IP1 significantly increased the number of EdU-positive cells and cell viability, and also upregulated mRNA expression of cell proliferation-associated genes including CCND1 and CDK2 in vitro cultured cells. Conversely, knockdown of PDZK1IP1 mediated by siRNA technique significantly inhibited subcutaneous preadipocyte proliferation and downregulated mRNA expression of cell proliferation-associated genes including CCNE1, CCND1 and CDK2. Collectively, these results suggested that PDZK1IP1 can promote proliferation of goat subcutaneous preadipocyte.

Knockdown of KLF7 inhibits the differentiation of both intramuscular and subcutaneous preadipocytes in goat.

KLF7 belongs to the Krüppel-like factors (KLFs) family, which function as transcriptional regulators controlling a number of basic cellular processes, involving proliferation, differentiation, and migration. Here, we reveal insights into the differentiated expression of KLF7 in different goat tissues and different stages of growth, and the inhibition role of KLF7 knockdown to differentiation by using goat intramuscular and subcutaneous preadipocytes. We demonstrate that KLF7 expression is obviously changed during the differentiation of preadipocytes into mature adipocytes. Knockdown of KLF7 inhibited lipid droplet accumulation, reduced the expression of adipogenic markers both in intramuscular and subcutaneous preadipocytes in goats, suggesting that KLF7 is a novel regulator of adipogenesis. KLF7 expression changed also up or down-regulation the other KLF family members, but there were differences between these two types of cells. Investigation into the mechanism that KLF7 regulates preadipocyte differentiation revealed that KLF family members KLF1, KLF5, KLF6, KLF8, KLF11, KLF12, KLF16, KLF17 and adipogenic markers C/EBPα and SREBP1 promoter region present KLF7 transcriptional binding sites. Altogether, the data here identify KLF7 as a novel regulator of adipogenesis.

FGF1 promotes the differentiation of goat intramuscular and subcutaneous preadipocytes.

Fibroblast growth factor 1(FGF1) has been proved to bind to specific signal molecules and activate intracellular signal transduction, leading to proliferation or differentiation of cells. However, the role of FGF1 in goat adipocytes is still unclear. Here, we investigated its role in lipogenesis of goats, which depends on the activation of FGFRs. In goat intramuscular and subcutaneous adipocytes, we observed that adipocytes accumulation was inhibited by interfering of FGF1, the expression of C/EBPα, C/EBPß, LPL, Pref-1, PPARγ, AP2, KLF4, KLF6, KLF10 and KLF17 were significantly down-regulated (p < 0.05). When the FGF1 was up-regulated, the opposite result was found, while the expression of C/EBPß, LPL, PPARγ, SREBP1, AP2, KLF4, KLF7, KLF15, KLF16 and KLF17 were increased significantly (p < 0.05) in goat intramuscular and subcutaneous adipocytes. The expression level of FGFR1 was significantly and decreased with the interference of FGF1, and increased with the overexpression of FGF1. But in goat subcutaneous adipocytes, only the expression of FGFR2 was consistent with the expression of FGF1. Interference methods confirmed that FGFR1 or FGFR2 and FGF1 have the similarly promoting function in adipocytes differentiation. With the co-transfection technology, we confirmed that FGF1 promoted the differentiation of intramuscular and subcutaneous adipocytes might via FGFR1 or FGFR2, respectively.

Effect of TEA domain transcription factor 1 (TEAD1) on the differentiation of intramuscular preadipocytes in goats.

TEA domain transcription factor 1 (TEAD1), also called TEF-1, acts as a transcriptional enhancer to regulate muscle-specific gene expression. However, the role of TEAD1 in regulating intramuscular preadipocyte differentiation in goats is unclear. The aim of this study was to obtain the sequence of TEAD1 gene and elucidate the effect of TEAD1 on goat intramuscular preadipocyte differentiation in vitro and its possible mechanism. The results showed that the goat TEAD1 gene CDS region sequence was 1311 bp. TEAD1 gene was widely expressed in goat tissues, with the highest expression in brachial triceps (p < 0.01). The expression of TEAD1 gene in goat intramuscular adipocytes at 72 h was extremely significantly higher than that at 0 h (p < 0.01). Overexpression of goat TEAD1 inhibited the accumulation of lipid droplets in goat intramuscular adipocyte. The relative expression of differentiation marker genes SREBP1, PPARγ, C/EBPß were significantly down-regulated (all p < 0.01), but PREF-1 was significantly up-regulated (p < 0.01). Binding analysis showed that there were multiple binding sites between the DNA binding domain of goat TEAD1 and the promoter binding region of SREBP1, PPARγ, C/EBPß and PREF-1. In conclusion, TEAD1 negatively regulates the differentiation of goat intramuscular preadipocytes.

The Important Role of m6A-Modified circRNAs in the Differentiation of Intramuscular Adipocytes in Goats Based on MeRIP Sequencing Analysis.

Intramuscular fat contributes to the improvement of goat meat quality. N6-Methyladenosine (m6A)-modified circular RNAs play important roles in adipocyte differentiation and metabolism. However, the mechanisms by which m6A modifies circRNA before and after differentiation of goat intramuscular adipocytes remain poorly understood. Here, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and circRNA sequencing (circRNA-seq) to determine the distinctions in m6A-methylated circRNAs during goat adipocyte differentiation. The profile of m6A-circRNA showed a total of 427 m6A peaks within 403 circRNAs in the intramuscular preadipocytes group, and 428 peaks within 401 circRNAs in the mature adipocytes group. Compared with the intramuscular preadipocytes group, 75 peaks within 75 circRNAs were significantly different in the mature adipocytes group. Furthermore, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of intramuscular preadipocytes and mature adipocytes showed that the differentially m6A-modified circRNAs were enriched in the PKG signaling pathway, endocrine and other factor-regulated calcium reabsorption, lysine degradation, etc. m6A-circRNA-miRNA-mRNA interaction networks predicted the potential m6A-circRNA regulation mechanism in different goat adipocytes. Our results indicate that there is a complicated regulatory relationship between the 12 upregulated and 7 downregulated m6A-circRNAs through 14 and 11 miRNA mediated pathways, respectively. In addition, co-analysis revealed a positive association between m6A abundance and levels of circRNA expression, such as expression levels of circRNA_0873 and circRNA_1161, which showed that m6A may play a vital role in modulating circRNA expression during goat adipocyte differentiation. These results would provide novel information for elucidating the biological functions and regulatory characteristics of m6A-circRNAs in intramuscular adipocyte differentiation and could be helpful for further molecular breeding to improve meat quality in goats.

Expression Variation of CPT1A Induces Lipid Reconstruction in Goat Intramuscular Precursor Adipocytes.

Intramuscular fat (IMF) deposition is one of the most important factors affecting meat quality and is closely associated with the expression of carnitine palmitoyl transferase 1A (CPT1A) which facilitates the transfer of long-chain fatty acids (LCFAs) into the mitochondria. However, the role of how CPT1A regulates the IMF formation remains unclear. Herein, we established the temporal expression profile of CPT1A during the differentiation of goat intramuscular precursor adipocytes. Functionally, the knockdown of CPT1A by siRNA treatment significantly increased the mRNA expression of adipogenic genes and promoted lipid deposition in goat intramuscular precursor adipocytes. Meanwhile, a CPT1A deficiency inhibited cell proliferation and promoted cell apoptosis significantly. CPT1A was then supported by the overexpression of CPT1A which significantly suppressed the cellular triglyceride deposition and promoted cell proliferation although the cell apoptosis also was increased. For RNA sequencing, a total of 167 differential expression genes (DEGs), including 125 upregulated DEGs and 42 downregulated DEGs, were observed after the RNA silencing of CPT1A compared to the control, and were predicted to enrich in the focal adhesion pathway, cell cycle, apoptosis and the MAPK signaling pathway by KEGG analysis. Specifically, blocking the MAPK signaling pathway by a specific inhibitor (PD169316) rescued the promotion of cell proliferation in CPT1A overexpression adipocytes. In conclusion, the expression variation of CPT1A may reconstruct the lipid distribution between cellular triglyceride deposition and cell proliferation in goat intramuscular precursor adipocyte. Furthermore, we demonstrate that CPT1A promotes the proliferation of goat adipocytes through the MAPK signaling pathway. This work widened the genetic regulator networks of IMF formation and delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Angiopoietin-like protein 8, molecular cloning and regulating lipid accumulation in goat intramuscular preadipocytes.

This study aimed to clone the full-length open reading frame (ORF) of goat ANGPTL8 gene sequence, reveal its molecular and expression characteristics, and explore its effect on the differentiation of goat intramuscular preadipocytes. The full-length ORF sequence of goat ANGPTL8 gene was cloned by RT-PCR technology, and bioinformatics analysis was performed by related biological software. RT-qPCR was used to detect the expression of ANGPTL8 mRNA in goat tissues. Further use of RNA interference to study the effect of ANGPTL8 on the differentiation of goat intramuscular preadipocytes. The total length of the ANGPTL8 gene nucleotide sequence is 717 bp, including 597 bp of ORF, encoding 198 amino acids. Goat ANGPTL8 has the closest relationship with sheep, it was widely expressed in different tissues, and relatively enriched in liver. The silence of ANGPTL8 inhibited the accumulation of lipid droplets by 5.76% in goat intramuscular preadipocytes (p > 0.05) and significantly suppressed the expression of the genes related to preadipocytes differentiation, fatty acid synthesis and transport (p＜0.05 or p＜0.01). These data illuminate the speculation that ANGPTL8 may involve in the lipid accumulation regulation via the control of PPARγ and C/EBPß in goat adipocytes.

Knockdown of adiponectin promotes the adipogenesis of goat intramuscular preadipocytes.

Intramuscular fat (IMF) content determined by the intramuscular preadipocytes differentiation has a huge influence on the sensory quality traits of meats. It was reported that the adiponectin (ADIPOQ) gene could promote adipocytes differentiation, but the underlying molecular and functional characterization of the ADIPOQ for regulating goat IMF deposition remained unknown. Herein, the knockdown of ADIPOQ was mediated by siRNAs during goat intramuscular preadipocytes differentiation. Also, the qRT-PCR technique was performed to detect the mRNA levels of target genes in multiply experiment groups. These results showed that the ADIPOQ was expressed more than ∼400 folds in subcutaneous adipose tissue compared to that of heart tissue, and the mRNA level of ADIPOQ reached a peak at Hour 60 during the differentiation process, while at Hour 36 did ADIPOR1 and ADIPOR2. Moreover, the knockdown of ADIPOQ promoted the intramuscular preadipocytes differentiation and accelerated the lipid accumulation in the mature adipocytes with down-regulating the ADIPOR1 and preadipocyte factor 1 (Pref-1) mRNA levels and up-regulating the mRNA expression levels of the CAAT/enhancer-binding proteins (C/EBPs) and transcription factor peroxisomal proliferator-activated receptor γ (PPARγ), etc. Our study will provide a new opposite insight that the inhibition of ADIPOQ expression during intramuscular preadipocytes differentiation promotes goat IMF deposition.

MiR-421 regulates goat intramuscular preadipocytes differentiation via targeting FGF13.

As a member of the MicroRNA s (miRNAs) family, miR-421 has been widely studied in regulating the proliferation and apoptosis of cancer cells a. However, there are still no reports on miR-421 in regulating adipocyte differentiation and its related mechanisms. Accordingly, this study aimed to investigate the potential involvement of miR-421 in goat intramuscular preadipocytes (P_IMA). The expression level of miR-421 was measured via quantitative real-time PCR during goat P_IMA differentiation. And the effects of miR-421 on goat P_IMA differentiation were studied by liposome transfection, Oil red O staining and qRT-PCR. Furthermore, the miR-421 target was searched and the underlying mechanism was clarified by luciferase reporter assay and rescue experiment. Our results showed that inhibition of miR-421 could accumulation of lipid droplets by upregulation the expression level of AP2, LPL, C/EBPα and SREBP1. Further studies showed that fibroblast growth factor 13 (FGF13) was the direct target of miR-421. Knocking down of FGF13 expression could inhibit lipid droplet formation and down-regulated the expression of key adipogenic regulatory genes. In addition, the rescue experiment revealed that FGF13 is involved in miR-421-induced differentiation of goat P_IMA as a key factor. Overall, these findings indicate that miR-421 is a negative regulator in the progression of differentiation of goat P_IMA by inhibiting the expression of FGF13.

Cloning of goat PGAM2 gene and its overexpression promotes the differentiation of intramuscular preadipocytes.

As a member of the PGAMs family, PGAM2 has been proved to catalyze the reversible reaction of 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA) in the glycolytic pathway. However, it is unclear whether PGAM2 has a role in regulating differentiation in goat intramuscular preadipocytes. Here, this study was carried to clone the open reading frame (ORF) of goat PGAM2, elucidate its molecular and expressional characteristics, and evaluate the involvement in adipogenesis of intramuscular preadipocytes. According to our findings, the cloned goat PGAM2 gene was 784 bp in full length, including 762 bp of ORF and encoding a protein of 253 amino acids. The expressional level of PGAM2 peaked at 48 hours after induced adipogenic differentiation and was highest in the skeletal muscle of triceps. Moreover, overexpression of PGAM2 transfected by its overexpression plasmid promotes lipid accumulation of goat intramuscular adipocyte as shown by Oil Red O and bodipy staining, accompanied by up-regulating the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) (p < 0.001), sterol regulatory element-binding protein 1 (SREBP1) (p < 0.001), CCAAT/Enhancer-binding protein α (C/EBPα) (p < 0.01) and lipoprotein lipase (LPL) (p < 0.01). Taken together, these findings indicate that PGAM2 is a positive regulator for goat intramuscular adipocytes and provide new insights into improvement intramuscular fat deposition in goat meat.

Chi-Circ_0006511 Positively Regulates the Differentiation of Goat Intramuscular Adipocytes via Novel-miR-87/CD36 Axis.

Goats are an important livestock and goat meat is essential to local people. The intramuscular fat (IMF) content has a great influence on the quality of goat meat. The intramuscular preadipocytes differentiation is closely related to the IMF deposition; however, its potential regulatory mechanisms remain unclear. CircRNAs were revealed to be involved in multiple biological progressions. In this study, we took primary goat intramuscular preadipocyte (GIMPA) as the study model to verify the function and mechanism of chi-circ_0006511, which was abundant and up-regulated in mature adipocytes (GIMA). The results showed that the expression level of chi-circ_0006511 gradually increased in the early stage of GIMPA differentiation, and chi-circ_0006511 was confirmed to promote GIMPA lipid droplets aggregation and up-regulate the adipogenic differentiation determinants, further promoting GIMPA differentiation. Mechanistically, chi-circ_0006511 exerts its function by sponging novel-miR-87, thereby regulating the expression of CD36. The results from this study provided novel significant information to better understand the molecular regulatory mechanism of intramuscular preadipocytes differentiation, thereby providing a new reference for the intramuscular fat adipogenesis in goats.

TMT-based quantitative proteomics analysis reveals the key proteins related with the differentiation process of goat intramuscular adipocytes.

BACKGROUND: Intramuscular adipocytes differentiation is a complex process, which is regulated by various transcription factor, protein factor regulators and signal transduction pathways. However, the proteins and signal pathways that regulates goat intramuscular adipocytes differentiation remains unclear. RESULT: In this study, based on nanoscale liquid chromatography mass spectrometry analysis (LC-MS/MS), the tandem mass tag (TMT) labeling analysis was used to investigate the differentially abundant proteins (DAPs) related with the differentiation process of goat intramuscular adipocytes. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the characterization of the identified DAPs. The candidate proteins were verified by parallel reaction monitoring analysis. As a result, a total of 123 proteins, 70 upregulation proteins and 53 downregulation proteins, were identified as DAPs which may be related with the differentiation process of goat intramuscular adipocytes. Furthermore, the cholesterol metabolism pathway, glucagon signaling pathway and glycolysis / gluconeogenesis pathway were noticed that may be the important signal pathways for goat Intramuscular adipocytes differentiation. CONCLUSIONS: By proteomic comparison between goat intramuscular preadipocytes (P_IMA) and intramuscular adipocytes (IMA), we identified a series protein that might play important role in the goat intramuscular fat differentiation, such as SRSF10, CSRP3, APOH, PPP3R1, CRTC2, FOS, SERPINE1 and AIF1L, could serve as candidates for further elucidate the molecular mechanism of IMF differentiation in goats.

Molecular and expression characteristics of resistin (RETN) and its effects on the differentiation of intramuscular preadipocyte in goat.

Resistin (RETN) is a hormone secreted by adipocytes, which plays an important role in glucose and lipid metabolism. The aim of this study is to clone and obtain the full length open reading frame (ORF) of goat RETN gene sequence, and to reveal its molecular and expression characteristics. Simultaneously, we explore its effect on the differentiation of intramuscular preadipocytes in goat. The full length ORF sequence of goat RETN gene was cloned by RT-PCR technique, and bioinformatics analysis was performed though relevant biological softwares. In this study, the expression of RETN mRNA in goat tissues and intramuscular preadipocytes during differentiation was detected by qPCR technique. Furthermore, RNA interference was used to explore the effects of RETN on intramuscular preadipocytes differentiation in goat. The results showed that the cloned goat RETN gene sequence was 428 bp in length, of which the ORF was 330 bp, encoding 109 amino acids. Sequence analysis revealed that it had 12 phosphorylation sites and an O-glycosylation site, and its protein contained a signal peptide sequence. Also, the RETN gene is expressed in goat various analyzed tissues, and the results showed that the expression of RETN gene in lung tissue was higher than that in other analyzed tissues of goat (p < .01). Moreover, the expression level of RETN gene in the goat's intramuscular preadipocytes decreased first and then increased, and reached the highest on the fifth day, which was significantly higher than that of undifferentiated intramuscular preadipocytes (p < .001). After transfecting intramuscular preadipocyte with siRNA, we found that the mRNA level of RETN was significantly down-regulated by 70% and 87% (p < .01). Oil red O staining results showed that the interference of RETN gene can promote the differentiation of intramuscular preadipocytes. After knockdown of RETN with siRNA, the PPARγ, AP2, C/EBPα, C/EBPß and SREBP1 genes were significantly up-regulated (p < .01). Thus, it can be inferred that RETN inhibits the differentiation of goat intramuscular preadipocytes, probably through regulating the expression of C/EBPα, C/EBPß, PPARγ, AP2 and SREBP-1 genes.

RXRα cooperates with KLF8 to promote the differentiation of intramuscular preadipocytes in goat.

Retinoid X receptor α (RXRα) is thought to be a key regulator in lipid metabolism, glucose metabolism, adipogenic differentiation, gene expression, and inflammatory response. However, it is not clear whether RXRα has any role in intramuscular preadipocyte of goat. In the current investigation, we report that adenovirus overexpression of RXRα promotes lipid accumulation in intramuscular adipocyte and up-regulates the expression of positive indicator genes (PPARγ and GLUT4). Next, knockdown of RXRα does not affect adipogenesis of intramuscular adipocyte. This phenomenon may be caused by the compensatory increase of RXRγ. In addition, we found that KLF8 mediates the positive role of RXRα. Knockdown of KLF8 by siRNA attenuated the effect of RXRα on adipocyte differentiation. Importantly, the promoter activity of KLF8 is enhanced when overexpression of RXRα. Taken together, our data suggest that RXRα promotes the differentiation of intramuscular preadipocytes in goat through targeting KLF8, but it could be replaced by RXRγ. These results provide new insights into the quality improvement of goat meat.