Porcine Preadipocyte Differentiation Research Articles

Effect of LncRNA LOC106505926 on myogenesis and Lipogenesis of porcine primary cells

BackgroundSkeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving the quality of carcass and meat. In the present study, whole transcriptome sequencing (including RNA-Seq and miRNA-Seq) was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age.ResultsThe results showed that a total of 245 differentially expressed miRNAs were screened in any two comparisons, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Pearson correlation coefficient analysis showed that long non-coding RNA (lncRNA) LOC106505926 and CXXC5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and CXXC5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of CXXC5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN.ConclusionsCollectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition.

Role of ADAR1 on Proliferation and Differentiation in Porcine Preadipocytes.

Recent research has identified ADAR1 as a participant in the regulation of lipid accumulation in mice. However, there are no reports on the roles of ADAR1 in proliferation, apoptosis and differentiation of porcine preadipocytes. In this study, we investigated the role of ADAR1 in differentiation, proliferation and apoptosis of porcine preadipocytes using CCK-8, EdU staining, cell cycle detection, RT-qPCR, Western blot, a triglyceride assay and Oil Red O staining. The over-expression of ADAR1 significantly promoted proliferation but inhibited the differentiation and apoptosis of porcine preadipocytes. The inhibition of ADAR1 had the opposite effect on the proliferation, differentiation and apoptosis of porcine preadipocytes with over-expressed ADAR1. Then, the regulation mechanisms of ADAR1 on preadipocyte proliferation were identified using RNA-seq, and 197 DEGs in response to ADAR1 knockdown were identified. The MAPK signaling pathway is significantly enriched, indicating its importance in mediating fat accumulation regulated by ADAR1. The study's findings will aid in uncovering the mechanisms that regulate fat accumulation through ADAR1.

MiR-10a-5p Regulates the Proliferation and Differentiation of Porcine Preadipocytes Targeting the KLF11 Gene.

In the swine industry, meat quality, color, and texture are influenced by the excessive differentiation of fat cells. miRNAs have emerged as integral regulators of adipose development. This study delves into the influence of miR-10a-5b on the proliferation and differentiation of pig preadipocytes. Our findings reveal that miR-10a-5b is prevalent across various tissues. It hinders preadipocyte proliferation, amplifies the expression of adipogenic genes, promotes lipid accumulation, and, as a result, advances preadipocyte differentiation. We predict that KLF11 is the target gene of miRNA. A dual-fluorescence reporter assay was conducted to validate the binding sites of miR-10a-5b on the 3'UTR of the KLF11 mRNA. Results showed that miR-10a-5b targeted KLF11 3'UTR and reduced the fluorescence activity of the dual-fluorescent reporter vector. Our research also indicates that miR-10a-5b targets and downregulates the expression of both mRNA and the protein levels of KLF11. During the differentiation of the preadipocytes, KLF11 inhibited adipose differentiation and was able to suppress the promotion of adipose differentiation by miR-10a-5b. This underscores miR-10a-5b's potential as a significant regulator of preadipocyte behavior by modulating KLF11 expression, offering insights into the role of functional miRNAs in fat deposition.

Integrated analysis strategy of genome-wide functional gene mining reveals DKK2 gene underlying meat quality in Shaziling synthesized pigs

BackgroundShaziling pig is a well-known indigenous breed in China who has superior meat quality traits. However, the genetic mechanism and genomic evidence underlying meat quality characteristics of Shaziling pigs are still unclear. To explore and investigate the germplasm characteristics of Shaziling pigs, we totally analyzed 67 individual’s whole genome sequencing data for the first time (20 Shaziling pigs [S], 20 Dabasha pigs [DBS], 11 Yorkshire pigs [Y], 10 Berkshire pigs [BKX], 5 Basha pigs [BS] and 1 Warthog).ResultsA total of 2,538,577 SNPs with high quality were detected and 9 candidate genes which was specifically selected in S and shared in S to DBS were precisely mined and screened using an integrated analysis strategy of identity-by-descent (IBD) and selective sweep. Of them, dickkopf WNT signaling pathway inhibitor 2 (DKK2), the antagonist of Wnt signaling pathway, was the most promising candidate gene which was not only identified an association of palmitic acid and palmitoleic acid quantitative trait locus in PigQTLdb, but also specifically selected in S compared to other 48 Chinese local pigs of 12 populations and 39 foreign pigs of 4 populations. Subsequently, a mutation at 12,726-bp of DKK2 intron 1 (g.114874954 A > C) was identified associated with intramuscular fat content using method of PCR–RFLP in 21 different pig populations. We observed DKK2 specifically expressed in adipose tissues. Overexpression of DKK2 decreased the content of triglyceride, fatty acid synthase and expression of relevant genes of adipogenic and Wnt signaling pathway, while interference of DKK2 got contrary effect during adipogenesis differentiation of porcine preadipocytes and 3T3-L1 cells.ConclusionsOur findings provide an analysis strategy for mining functional genes of important economic traits and provide fundamental data and molecular evidence for improving pig meat quality traits and molecular breeding.

Effects of HOXC8 on the Proliferation and Differentiation of Porcine Preadipocytes.

Transcription factor Homeobox C8 (HOXC8) is identified as a white adipose gene as revealed by expression profile analysis in fat tissues. However, the specific role of HOXC8 in fat accumulation remains to be identified. This study was designed to reveal the effects of HOXC8 on preadipocyte proliferation and differentiation. We first make clear that the expression of HOXC8 is associated with fat contents in muscles, highlighting a role of HOXC8 in fat accumulation. Next, it is demonstrated that HOXC8 promotes the proliferation and differentiation of preadipocytes through gain- and loss-of-function assays in primary cultured porcine preadipocytes. Then, mechanisms underlying the regulation of HOXC8 on preadipocyte proliferation and differentiation are identified with RNA sequencing, and a number of differentially expressed genes (DEGs) in response to HOXC8 knockdown are identified. The top GO (Gene Ontology) terms enriched by DEGs involved in proliferation and differentiation, respectively, are identical. IL-17 signaling pathway is the common one significantly enriched by DEGs involved in preadipocyte proliferation and differentiation, respectively, indicating its importance in mediating fat accumulation regulated by HOXC8. Additionally, we find that the inhibition of proliferation is one of the main processes during preadipocyte differentiation. The results will contribue to further revealing the mechanisms underlying fat accumulation regulated by HOXC8.

The Effects of Neuropeptide B on Proliferation and Differentiation of Porcine White Preadipocytes into Mature Adipocytes

Neuropeptide B (NPB) affects energy homeostasis and metabolism by binding and activating NPBWR1 and NPBWR2 in humans and pigs. Recently, we reported that NPB promotes the adipogenesis of rat white and brown preadipocytes as well as 3T3-L1 cells. In the present study, we evaluated the effects of NPB on the proliferation and differentiation of white porcine preadipocytes into mature adipocytes. We identified the presence of NPB, NPBWR1, and NPBWR2 on the mRNA and protein levels in porcine white preadipocytes. During the differentiation process, NPB increased the mRNA expression of PPARγ, C/EBPβ, C/EBPα, PPARγ, and C/EBPβ protein production in porcine preadipocytes. Furthermore, NPB stimulated lipid accumulation in porcine preadipocytes. Moreover, NPB promoted the phosphorylation of the p38 kinase in porcine preadipocytes, but failed to induce ERK1/2 phosphorylation. NPB failed to stimulate the expression of C/EBPβ in the presence of the p38 inhibitor. Taken together, we report that NPB promotes the differentiation of porcine preadipocytes via a p38-dependent mechanism.

CircHOMER1 inhibits porcine adipogenesis via the miR-23b/SIRT1 axis.

Fat deposition is critical to pork quality. However, the mechanism of fat deposition remains to be elucidated. Circular RNAs (circRNAs) are ideal biomarkers and are involved in adipogenesis. Here, we investigated the effect and mechanism of circHOMER1 on porcine adipogenesis in vitro and in vivo. Western blotting, Oil red O staining, and HE staining were used to assess the function of circHOMER1 in adipogenesis. The results showed that circHOMER1 inhibited adipogenic differentiation of porcine preadipocytes and suppressed adipogenesis in mice. Dual-luciferase reporter gene, RIP, and pull-down assays demonstrated that miR-23b directly bound to circHOMER1 and the 3'-UTR of SIRT1. Rescue experiments further illustrated the regulatory relationship among circHOMER1, miR-23b, and SIRT1. Conclusively, we demonstrate that circHOMER1 plays an inhibitory role in porcine adipogenesis through miR-23b and SIRT1. The present study revealed the mechanism of porcine adipogenesis, which may be helpful to improve pork quality.

MiR-26a-5p Regulates Adipocyte Differentiation via Directly Targeting ACSL3 in Adipocytes

ABSTRACT Preadipocytes become mature adipocytes after proliferation and differentiation, and although many genes and microRNAs have been identified in intramuscular fat, their physiological function and regulatory mechanisms remain largely unexplored. miR-26a-5p has been reported to be related to fat deposition, but its effect on porcine preadipocyte differentiation has not been explored. In this study, bioinformatics analysis and luciferase reporter assay identified that miR-26a-5p binds to the 3ʹUTR of Acyl-CoA synthetase long-chain family member 3 (ACSL3) mRNA. The model for porcine intramuscular preadipocyte differentiation was established to explore the function of miR-6a-5p-ACSL3 on adipocyte differentiation. ACSL3 knockdown markedly reduced the triglycerides (TG) content of cells, as well as the mRNA levels of adipogenic marker genes (PPAR-γ and SREBP-1c). The number of lipid droplets in cells transfected with a miR-26a-5p mimic is significantly reduced, consistent with ACSL3 knockdown results, while the miR-26a-5p inhibitor resulted in opposite results. Taken together, miR-26a-5p is a repressor of porcine preadipocyte differentiation and plays a vital role in ACSL3-mediated adipogenesis.

LncPLAAT3-AS Regulates PLAAT3-Mediated Adipocyte Differentiation and Lipogenesis in Pigs through miR-503-5p.

Animal fat deposition has a significant impact on meat flavor and texture. However, the molecular mechanisms of fat deposition are not well understood. LncPLAAT3-AS is a naturally occurring transcript that is abundant in porcine adipose tissue. Here, we focus on the regulatory role of lncPLAAT3-AS in promoting preadipocyte proliferation and adipocyte differentiation. By overexpressing or repressing lncPLAAT3 expression, we found that lncPLAAT3-AS promoted the transcription of its host gene PLAAT3, a regulator of adipocyte differentiation. In addition, we predicted the region of lncPLAAT3-AS that binds to miR-503-5p and showed by dual luciferase assay that lncPLAAT3-AS acts as a sponge to absorb miR-503-5p. Interestingly, miR-503-5p also targets and represses PLAAT3 expression and helps regulate porcine preadipocyte proliferation and differentiation. Taken together, these results show that lncPLAAT3-AS upregulates PLAAT3 expression by absorbing miR-503-5p, suggesting a potential regulatory mechanism based on competing endogenous RNAs. Finally, we explored lncPLAAT3-AS and PLAAT3 expression in adipose tissue and found that both molecules were expressed at significantly higher levels in fatty pig breeds compared to lean pig breeds. In summary, we identified the mechanism by which lncPLAAT3-AS regulates porcine preadipocyte proliferation and differentiation, contributing to our understanding of the molecular mechanisms of lipid deposition in pigs.

Caveolae‐associated protein 3 promotes adipogenic differentiation of porcine preadipocytes by promoting extracellular signal‐regulated kinase phosphorylation

Fat deposition is one of the key factors affecting the economic development of pig husbandry. The aim of this study was to investigate the expression characteristics of caveolae-associated protein 3 (CAVIN3) and to elucidate its effect and mechanism on adipogenic differentiation of porcine preadipocytes. Cell transfection, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and oil red O staining were used to detect the effect of CAVIN3 on the differentiation of porcine preadipocytes. The results showed that CAVIN3 was expressed in various tissues, with higher expression in adipose tissue, differentially expressed during cell adipogenic differentiation, and mainly distributed in the cytoplasm. Functional studies showed that, after CAVIN3 interference in preadipocytes, the expression of adipogenic factors and the content of lipid droplets were significantly decreased (p < 0.05). The results were reversed after CAVIN3 was overexpressed. The mechanism research showed that LY3214996 inhibited the extracellular signal-regulated kinase (ERK) phosphorylation and further inhibited lipogenic factors expression. Overexpression of CAVIN3 attenuates the inhibitory effect of LY3214996 on ERK phosphorylation and attenuates its inhibitory effect on adipogenic differentiation. Therefore, this study demonstrated that CAVIN3 promotes the differentiation of porcine preadipocytes by promoting ERK phosphorylation. The present study can lay a theoretical foundation for further studying the molecular mechanism of porcine fat deposition.

The long non-coding RNA lncMYOZ2 mediates an AHCY/MYOZ2 axis to promote adipogenic differentiation in porcine preadipocytes

Long non-coding RNAs (lncRNAs) play a vital role in regulating adipogenesis. However, the associated regulatory mechanisms have yet to be described in detail in pig. In this study, we demonstrate a critical role for lncMYOZ2 in adipogenesis from porcine preadipocytes. Specifically, lncMYOZ2 was more abundant in the adipose tissue of Mashen (fat-type) pigs than for Large White (lean-type) pigs, and knockdown of this lncRNA significantly inhibited the differentiation of porcine preadipocytes into adipocytes. Mechanistically, we used RNA pull-down and RIP assays to establish that lncMYOZ2 interacts with adenosylhomocysteinase (AHCY). Moreover, lncMYOZ2 knockdown increased promoter methylation of the target gene MYOZ2 and lowered its expression. Finally, we describe a positive regulatory role for MYOZ2 in adipogenesis. Collectively, these findings establish lncMYOZ2 as an important epigenetic regulator of adipogenesis via the aforementioned AHCY/MYOZ2 pathway, and provide insights into the role of lncRNAs in porcine adipose development.

Genome-wide characterization of lncRNAs and mRNAs in muscles with differential intramuscular fat contents.

Meat quality is one of the most important traits in pig production. Long non-coding RNAs (lncRNAs) have been involved in diverse biological processes such as muscle development through regulating gene expression. However, studies on lncRNAs lag behind and a comparatively small number of lncRNAs have been identified in pigs. Also, the effects of lncRNAs on meat quality remain to be characterized. Here, we analyzed lncRNAs in longissimus thoracis (LT) and semitendinosus (ST) muscles, being different in meat quality, with RNA-sequencing technology. A total of 500 differentially expressed lncRNAs (DELs) and 2,094 protein-coding genes (DEGs) were identified. Through KEGG analysis on DELs, we first made clear that fat deposition might be the main reason resulting in the differential phenotype of LT and ST, for which cGMP–PKG and VEGF signaling pathways were the most important ones. In total, forty-one key DELs and 50 DEGs involved in the differential fat deposition were then characterized. One of the key genes, cAMP-response element binding protein 1, was selected to confirm its role in porcine adipogenesis with molecular biology methods and found that it promotes the differentiation of porcine preadipocytes, consistent with its higher expression level and intramuscular fat contents in LT than that in ST muscle. Furthermore, through integrated analysis of DELs and DEGs, transcription factors important for differential fat deposition were characterized among which BCL6 has the most target DEGs while MEF2A was targeted by the most DELs. The results provide candidate genes crucial for meat quality, which will contribute to improving meat quality with molecular-breeding strategies.

MiR-23b Promotes Porcine Preadipocyte Differentiation via SESN3 and ACSL4.

Genetic improvement of pork quality is one of the hot topics in pig germplasm innovation. Backfat thickness and intramuscular fat content are important indexes of meat quality. MiRNAs are becoming recognized as a crucial regulator of adipose development. Therefore, it is crucial to understand how miR-23b regulates fat metabolism at the molecular level. In the present study, Oil Red O staining, and Western blot were used to evaluate the effect of miR-23b on the differentiation of porcine preadipocytes. Dual-luciferase reporter gene assay, pulldown, and RIP were used to reveal the mechanism of miR-23b regulating cell differentiation. The findings demonstrated that miR-23b promotes the expression of adipogenic factors and increases the content of lipid droplets, thus promoting the differentiation of preadipocytes. Further research found that miR-23b can directly bind to the 3’UTR of SESN3 to regulate adipogenic differentiation. In addition, it was speculated that miR-23b controls cell differentiation by positively regulating the expression of ACSL4 in other ways. Here, we demonstrate that miR-23b promotes the differentiation of porcine preadipocytes by targeting SESN3 and promoting the expression of ACSL4. The present study is meaningful to the improvement of pork quality and the development of animal husbandry.

C/EBPα promotes porcine pre-adipocyte proliferation and differentiation via mediating MSTRG.12568.2/FOXO3 trans-activation for STYX

As a key adipogenic marker, C/EBPα (CCAAT/enhancer binding protein α) is also an important factor in regulating targets containing CCAAT element for transcription, whose products include coding and non-coding RNAs (ncRNAs). However, knowledge of the mechanism of C/EBPα affecting pre-adipocyte proliferation and adipogenesis through regulating ncRNA is still limited. In this study, we firstly conducted an investigation concerning the impact of C/EBPα knockdown on porcine pre-adipocytes by using RNA sequencing (RNA-Seq) to identify the role of key ncRNAs, especially lncRNAs and their correlated mRNAs in regulating proliferation and differentiation of porcine pre-adipocytes. 97 differentially expressed (DE) mRNAs and 4 DE lncRNAs were identified in si-C/EBPα groups compared with the si-NC groups. Meanwhile, we found C/EBPα directly target the promoter of a novel lncRNA, namely MSTRG.12568.2, which was trans-correlated with STYX (serine/threonine/tyrosine interacting protein), an important candidate gene for regulating cell proliferation. Moreover, FOXO3 (forkhead box O3) was identified as a co-regulator with MSTR.12568.2 for STYX. Overexpression and knockdown of any of the MSTRG.12568.2, STYX, and FOXO3 increased and decreased the levels of pre-adipocyte proliferation and differentiation, respectively, which demonstrated that they played a positive role in adipogenesis of pre-adipocytes. Furthermore, our results revealed that FOXO3 was necessary for MSTRG.12568.2 to trans-activate STYX. We revealed that C/EBPα regulated pre-adipocyte proliferation and differentiation through mediating trans-activation of MSTRG.12568.2-FOXO3 to STYX. These results provide a novel regulation signal for C/EBPα to influence porcine pre-adipocyte proliferation and differentiation and greatly benefit to our understanding of molecular mechanism regulating subcutaneous adipogenesis.

Characterization of a Read-through Fusion Transcript, BCL2L2-PABPN1, Involved in Porcine Adipogenesis.

cis-Splicing of adjacent genes (cis-SAGe) has been involved in multiple physiological and pathological processes in humans. However, to the best of our knowledge, there is no report of cis-SAGe in adipogenic regulation. In this study, a cis-SAGe product, BCL2L2–PABPN1 (BP), was characterized in fat tissue of pigs with RT-PCR and RACE method. BP is an in-frame fusion product composed of 333 aa and all the functional domains of both parents. BP is highly conserved among species and rich in splicing variants. BP was found to promote proliferation and inhibit differentiation of primary porcine preadipocytes. A total of 3074/44 differentially expressed mRNAs (DEmRs)/known miRNAs (DEmiRs) were identified in porcine preadipocytes overexpressing BP through RNA-Seq analysis. Both DEmRs and target genes of DEmiRs were involved in various fat-related pathways with MAPK and PI3K-Akt being the top enriched. PPP2CB, EGFR, Wnt5A and EHHADH were hub genes among the fat-related pathways identified. Moreover, ssc-miR-339-3p was found to be critical for BP regulating adipogenesis through integrated analysis of mRNA and miRNA data. The results highlight the role of cis-SAGe in adipogenesis and contribute to further revealing the mechanisms underlying fat deposition, which will be conductive to human obesity control.

MiR-218-5p Affects Subcutaneous Adipogenesis by Targeting ACSL1, a Novel Candidate for Pig Fat Deposition

As a centre enzyme in fatty acid activation, acyl-CoA synthetase long-chain family member 1 (ACSL1) plays an important role in body lipid homeostasis. However, the functions of ACSL1 in the subcutaneous adipogenesis of pigs are largely unknown. In the present study, we found that the expression of ACSL1 significantly increased during the process of porcine preadipocyte differentiation. Moreover, silencing of ACSL1 in preadipocytes decreased levels of triglyceride and adipogenic-related markers, including FABP4, APOE, and FASN (p < 0.01), and simultaneously increased levels of lipolytic-related markers, such as ATGL and HSL (p < 0.05). Conversely, overexpression of ACSL1 in preadipocytes increased levels of triglyceride and FABP4, APOE, and FASN (p < 0.01), and reduced levels of ATGL and HSL (p < 0.05). Luciferase reporter assays revealed that ACSL1 is a target of miR-218-5p, which can reduce the mRNA and protein levels of ACSL1 by directly binding the 3′ untranslated region of ACSL1. Furthermore, miR-218-5p has an inhibition role in porcine preadipocyte differentiation by suppressing ACSL1 expression. Taken together, these data provide insights into the mechanism of the miR-218-5p/ACSL1 axis in regulating subcutaneous fat deposition of pigs.

Dietary dibutyryl cAMP supplementation regulates the fat deposition in adipose tissues of finishing pigs via cAMP/PKA pathway

This study investigated potential mechanism of dibutyryl-cAMP (db-cAMP) on porcine fat deposition. (1) Exp.1, 72 finishing pigs were allotted to 3 treatments (0, 10 or 20 mg/kg dbcAMP) with 6 replicates. dbcAMP increased the hormone sensitive lipase (HSL) activity and expression of β-adrenergic receptor (β-AR) and growth hormone receptor (GHR), but decreased expression of peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) and adipocyte fatty acid binding protein (A-FABP) in back fat. dbcAMP upregulated expression of β-AR, GHR, PPAR-γ2 and A-FABP, but decreased insulin receptor (INSR) expression in abdominal fat. Dietary dbcAMP increased HSL activity and expression of G protein-coupled receptor (GPCR), cAMP-response element-binding protein (CREB) and insulin-like growth factor-1 (IGF-1), but decreased fatty acid synthase (FAS) and lipoprotein lipase (LPL) activities, and expression of INSR, cAMP-response element-binding protein (C/EBP-α) and A-FABP in perirenal fat. (2) Exp. 2, dbcAMP suppressed the proliferation and differentiation of porcine preadipocytes in a time- and dose-dependent manner, which might be associated with increased activities of cAMP and protein kinase A (PKA), and expression of GPCR, β-AR, GHR and CREB via inhibiting C/EBP-α and PPAR-γ2 expression. Collectively, dbcAMP treatment may reduce fat deposition by regulating gene expression related to adipocyte differentiation and fat metabolism partially via cAMP-PKA pathway.

RNF20 affects porcine adipocyte differentiation via regulation of mitotic clonal expansion.

ObjectivesRNF20 is recognized as a main E3 ligase for monoubiquitination of histone H2B at lysine 120 (H2Bub). The critical role of RNF20 and H2Bub in various molecular events, such as DNA replication, RNA transcription, and DNA damage response, has been widely investigated and documented. However, its role in porcine adipogenesis remains unknown. In this study, we aimed to clarify the effect of RNF20 on porcine preadipocyte differentiation.Materials and MethodsBackfat tissues from fat‐type pigs (Bama and Meishan) and lean‐type pigs (Yorkshire and Landrace) were collected to detect the expression level of RNF20. Preadipocytes were isolated from Bama piglets and induced to differentiation. Small interfering RNAs were applied to deplete RNF20. Oil Red O staining, quantitative real‐time PCR, RNA‐seq, Western blot analysis, and EdU assays were performed to study the regulatory mechanism of RNF20 during adipogenesis.ResultsWe found that the expression levels of RNF20 and H2Bub were significantly higher in backfat tissues from fat‐type pigs than in those from lean‐type pigs. Consistently, the significantly induced expression of RNF20 and H2Bub was also observed in porcine differentiated adipocytes. In addition, knockdown of RNF20 greatly inhibited porcine adipogenesis, as evidenced by dramatically decreased lipid droplet formation and lower expression levels of adipogenic transcription masters in RNF20 knockdown cells. Mechanistically, the depletion of RNF20 decreases the cell proliferation and the level of p‐C/EBPβ via the Ras‐Raf‐MEK1/2‐ERK1/2 cascade pathway at the mitotic clonal expansion phase and therefore suppresses cell differentiation.ConclusionsOur results demonstrate that RNF20 is required for porcine preadipocyte differentiation.

TGF-β1-Mediated FDNCR1 Regulates Porcine Preadipocyte Differentiation via the TGF-β Signaling Pathway.

Simple SummaryFat differentiation affects lipid deposition and is a complex metabolic process. It has been previously reported that multiple transcription factors regulate adipocyte formation. With the in-depth study of epigenetics, in recent years it has been reported that long noncoding RNA (lncRNA) can effectively affect the formation of lipid droplets and thus regulate fat deposition. lncRNA can regulate cell function through a variety of mechanisms, the most studied is the mechanism of action of lncRNA as a miRNA molecular sponge. The purpose of this article is to explore the role of transforming growth factor-beta (TGF-β1) mediated lncRNA in the formation of porcine adipocytes, from the perspective of lncRNA to reveal the effect of TGF-β1 on the differentiation of porcine adipocytes, and provide a new way to improve the quality of pork.Adipocyte differentiation and lipid metabolism have important regulatory effects on the quality of meat from livestock. A variety of transcription factors regulate preadipocyte differentiation. Several studies have revealed that transforming growth factor-beta (TGF-β1) may play a key role in epithelial–mesenchymal transition (EMT); however, little is known about the effects of TGF-β1 treatment on porcine preadipocytes. To explore the role of TGF-β1 in porcine adipocyte differentiation, porcine preadipocytes were treated with 10 ng/mL TGF-β1, and two libraries were constructed for RNA-seq. We chose an abundant and differentially expressed long noncoding RNA (lncRNA), which we named fat deposition-associated long noncoding RNA1 (FDNCR1), for further study. RT-qPCR was used to detect mRNA levels of genes related to adipocyte differentiation. Triglyceride assay kits were used to detect lipid droplet deposition. TGF-β1 significantly suppressed porcine preadipocyte differentiation. We identified 8158 lncRNAs in total and 39 differentially expressed lncRNAs. After transfection with FDNCR1 siRNA, the mRNA expression of aP2, C/EBPα, and PPARγ and triglyceride levels significantly increased. Transfection with FDNCR1 siRNA significantly decreased protein levels of p-Smad2/Smad2 and p-Smad3/Smad3. These results demonstrate that FDNCR1 suppresses porcine preadipocyte differentiation via the TGF-β signaling pathway.

Upregulated microRNA-106a Promotes Porcine Preadipocyte Proliferation and Differentiation by Targeting Different Genes.

Adipose tissue is one of the main organs for the energy storage and supply of organisms. Adipose deposition and metabolism are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms. Previous studies have also shown that miR-106a plays a considerable role in the development of organisms. The regulatory mechanism of miR-106a on porcine preadipocytes is still not clear. In this study, preadipocytes were isolated from the neck subcutaneous deposits of 3–5-day old Chinese native Guanzhong black pigs using 5-ethynyl-20-deoxyuridine (EdU) staining and a CCK-8 assay to detect the number of proliferous cells and real-time qPCR (RT-qPCR) and western blot analysis to detect gene expression, as well as Oil Red O and BODIPY staining dye lipid droplets and flow cytometry (FCM) to detect cell cycles. We also used the double luciferase method to detect the relative luciferase activities. Upregulated miR-106a increased the number of proliferous cells and enhanced the expression of cell proliferation-related genes in porcine adipocytes. The double luciferase reporter vector confirmed that p21 was a target gene of miR-106a in the cell proliferation phase. miR-106a upregulation increased the number of lipid droplets and the expression of lipogenic genes and directly targeted BMP and activin membrane-bound inhibitor (BAMBI) in the process of differentiation. Our results indicated that miR-106a promotes porcine preadipocyte proliferation and differentiation by targeting p21 and BAMBI.