MiR-145 reduces the activity of PI3K/Akt and MAPK signaling pathways and inhibits adipogenesis in bovine preadipocytes.

Adipose tissue is the largest metabolic organ because of adipogenesis controlled by numerous miRNAs. MiR-145 is classified into the same cluster with famous miR-143. However, few studies have investigated the role of miR-145 in adipogenesis. In the current study, we observed that the expression of miR-145 was downregulated during bovine adipogenesis in vivo and in vitro. The results of RNA-Seq analysis showed that miR-145 mainly disturb the PI3K/Akt and MAPK signaling pathways in bovine preadipocytes. MiR-145 inhibited bovine preadipocyte differentiation and downregulated phosphorylation level of Akt and ERK1/2 proteins. Furthermore, insulin, as a powerful inducer initiating adipogenesis and an activator of the PI3K/Akt and MAPK signaling pathways, was able to rescue the downregulation of Akt and ERK1/2 phosphorylation levels caused by miR-145. Taken together, our findings suggest that miR-145 is a potent inhibitor of adipogenesis that may function by reducing the activity of PI3K/Akt and MAPK signaling pathways.

miR-424 Promotes Bovine Adipogenesis Through an Unconventional Post-Transcriptional Regulation of STK11.

Adipose tissue is the largest energy reservoir and secretory organ in the animal body, and is essential for maintaining normal physiological functions and metabolic balance. MicroRNAs regulate the process of adipogenic differentiation through post-transcriptional regulatory mechanisms. In the present study, miR-424 was upregulated during bovine adipocyte differentiation both in vivo and in vitro. The overexpression and interference of miR-424 exhibited the positive regulatory role in the differentiation of bovine adipocytes. Furthermore, miR-424 directly binds to the three prime untranslated region (3' UTR) of serine/threonine kinase 11 (STK11, also called LKB1), a master upstream gene in the AMP-activated protein kinase (AMPK) cascade, and up-regulates its expression. Functional studies showed that the knockdown of STK11 attenuated the pro-adipogenic effect of miR-424. Post-transcriptional regulation of STK11 by miR-424 was mediated potentially in an RNA binding protein (RBP) binding site-dependent manner. In conclusion, our study shows that miR-424 promotes bovine adipogenesis through an unconventional post-transcriptional regulation of STK11, which may serve as a potential target for the regulation of bovine adipogenesis and the improvement of livestock breeding efficiency.

Bta-miR-149-5p inhibits proliferation and differentiation of bovine adipocytes through targeting CRTCs at both transcriptional and posttranscriptional levels.

MicroRNAs are small, single stranded, and noncoding RNAs that have been proven to be potent regulators of adipogenesis. However, the role of bta-miR-149-5p in regulating bovine adipogenesis is still unclear. Expression profiling in different stages of adipogenesis revealed that bta-miR-149-5p was enriched in the proliferation stage, and also on Day 9 of differentiation in bovine adipocytes. Our gain of function study showed that bta-miR-149-5p can negatively regulate both bovine adipocyte proliferation and differentiation. Overexpression of bta-miR-149-5p suppressed the expression of proliferation marker genes at both the messenger RNA (mRNA) and protein levels, markedly decreased the percentage of S-phase cells, decreased the number of EdU-stained cells, and substantially reduced adipocyte proliferation vitality in the cell count assay. Collectively, these findings elucidated that bta-miR-149-5p inhibits adipocyte proliferation. Furthermore, overexpression of bta-miR-149-5p also suppressed the expression of adipogenic genes at both the mRNA and protein levels, inhibited lipid accumulation, and reduced the secretion of adiponectin in bovine adipocytes. Furthermore, a luciferase activity assay explored how bta-miR-149-5p targeted CRTCs (CRTC1 and CRTC2) directly. This targeting was further validated by the mRNA and protein level expression of CRTC1 and CRTC2, which were down regulated by bta-miR-149-5p overexpression. Moreover, bta-miR-149-5p indirectly targeted CRTC1 and CRTC2 through regulating their key transcription factors. Overexpression of bta-miR-149-5p suppressed the expression of SMAD3, while enriched the expression of NRF1, which are the key transcription factors and proven regulators of CRTC1. Overexpression of bta-miR-149-5p also repressed the expression of C/EBPγ, XBP1, INSM1, and ZNF263, which are the key regulators of CRTCs, at both the mRNA and protein levels. These findings suggest that bta-miR-149-5p is a negative regulator of CRTC1 and CRTC2 both at transcriptional and posttranscriptional level. Taken together, these findings suggest that bta-miR-149-5p can regulate adipogenesis, which implies that bta-miR-149-5p could be a target for increasing intramuscular fat in beef cattle.

Effect of ELOVL6 on the lipid metabolism of bovine adipocytes.

This study investigated the effect of ELOVL6 (elongation of very long chain fatty acids protein 6) and its underlying mechanism on lipid metabolism in bovine adipocytes. The ELOVL6 gene was overexpressed in bovine adipocytes by adenoviruses, and RNA sequencing was performed. Overexpression of ELOVL6 showed reduced proportions of C14:0 (Myristic) and C16:0 (palmitate) fatty acids and increased proportions of C18.0 (stearate) and C20:4n6 (arachidonic) fatty acids in adipocytes. In addition, a total of 2170 differentially expressed genes (DEGs) were found, containing 1802 up-regulated and 368 down-regulated genes. KEGG pathway analysis revealed that the down-regulated genes were linked with the regulation of lipolysis and the Wnt signaling pathway. The up-regulated genes were mainly involved in the FoxO signaling pathway; the PI3K-Akt signaling pathway; and the cAMP signaling pathway. In conclusion, our results suggest that ELOVL6 could affect the fatty acid composition in bovine adipocytes. We identified numerous related DEGs and pathways, which may provide a basis for studying the function and molecular mechanism of the ELOVL6 gene in regulating lipid metabolism.

RNA-seq reveal role of bovine TORC2 in the regulation of adipogenesis.

Low intramuscular adipose tissue (marbling) continues to be challenge for improving beef quality in Chinese cattle. Highly marbled meat is very desirable; hence, methods to increase IMF content have become a key aspect of improving meat quality. Therefore, research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality. This study investigated the effect of TORC2 and its underlying mechanism on lipid metabolism in bovine adipocytes. The TORC2 gene was downregulated in bovine adipocytes by siRNA, and RNA sequencing was performed. Downregulation of TORC2 negatively affected bovine adipocyte differentiation. In addition, a total of 577 DEGs were found, containing 146 up-regulated and 376 down-regulated genes. KEGG pathway analysis revealed that the DEGs were linked with neuroactive ligand-receptor interaction pathway, calcium signaling pathway, cAMP pathway, chemokine signaling pathway and Wnt signaling pathway. Gene Ontology (GO) term analysis of the DEGs showed that down-regulation of TORC2 gene significantly suppressed the genes regulating important GO terms of adipogenesis-related processes in bovine adipocytes, especially regulation of biological activity, regulation of primary metabolic process, regulation of multicellular organismal process, cell adhesion, lipid metabolic process, secretion, chemical homeostasis, regulation of transport, cell-cell signaling, cAMP metabolic process, cellular calcium ion homeostasis, fat cell differentiation, and cell maturation. In conclusion, our results suggest that TORC2 at least in part regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the TORC2 gene in regulating adipogenesis.

Copy number variation detection in Chinese indigenous cattle by whole genome sequencing.

Copy number variation (CNV) refers to a kind of structural variation, having functional and evolutionary effects on phenotypes. Thus far, further elucidation of the CNVs in different Chinese indigenous cattle breeds by whole genome sequencing have yet not been done. In this study, a comprehensive genomic analysis was performed on 75 cattle individuals including six Chinese indigenous cattle breeds and two non-native specialized beef cattle breeds. Based on the 11,486 CNVRs discovered, population analysis was performed, showed that all the cattle breeds clustered in to three clades, consistent with their lineages Bos taurus, Bos taurus × Bos indicus and Bos indicus. Importantly, a set of CNVRs related genes were found to be associated with the traits of interest, which include meat production or quality (CAST, ACTC1, etc.), adaption (BLA-DQB, EGLN2, etc.) and coat color (KIT, MITF, etc.). These results provide valuable full genome variation resources for Chinese bovine genome research and would be helpful for cattle breeding and selection programs in the future.

GR and Foxa1 promote the transcription of ANGPTL4 in bovine adipocytes.

ANGPTL4 (angiopoietin-like 4) is a secreted protein involved in triacylglycerol homeostasis. It is a key enzyme in lipolysis, which stimulates the oxidation of fatty acids and inhibits fat accumulation by inhibiting the activity of lipoprotein lipase (LPL). Using quantitative Real-Time PCR (qRT-PCR) to investigate the mRNA expression pattern of the bovine ANGPTL4 gene in different tissues and organs, we found that bovine ANGPTL4 had the highest expression level in the liver followed by subcutaneous adipose tissue. To clarify the molecular mechanism involved in the regulation of bovine ANGPTL4, we identified the transcriptional start site (TSS) of the ANGPTL4 gene and obtained 2011 bp of the 5' regulatory region. A series of 5' deletion promoter luciferase reporter assays revealed that the minimum functional promoter region of bovine ANGPTL4 was located at -568 bp to -261 bp relative to TSS. Two transcription factors, GR and Foxa1, were identified and considered as important transcriptional activators of ANGPTL4 by mutational analysis and RNA interference assays in combination with electrophoretic mobility shift assays (EMSA) in bovines. In conclusion, GR and Foxa1 were determined to be responsible for the regulation of ANGPTL4 transcription. Our results may provide a basis for further investigation of ANGPTL4 regulation and a reference for improvement of beef quality in cattle.

Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes: Roles of C/EBP, XBP1, INSM1 and ZNF263.

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2'-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5'UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5'UTR flanking region. The core promoter region of the TORC2 gene was identified at location -314 to -69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.

Transcriptional regulation of bovine elongation of very long chain fatty acids protein 6 in lipid metabolism and adipocyte proliferation.

The elongation of very long chain fatty acids protein 6 (ELOVL6) gene encodes a key enzyme that plays a role in lipogenesis through the catalytic elongation of both saturated and monounsaturated fatty acids. Previous studies have described the high expression of bovine ELOVL6 in adipose tissues. However, transcriptional regulation and the functional role of ELOVL6 in lipid metabolism and adipocyte proliferation remain unexplored. Here, a 1.5 kb fragment of the 5'-untranslated region promoter region of ELOVL6 was amplified from the genomic DNA of Qinchuan cattle and sequenced. The core promoter region was identified through unidirectional 5'-end deletion of the promoter plasmid vector. In silico analysis predicted important transcription factors that were then validated through site-directed mutation and small interfering RNA interference with an electrophoretic mobility shift assay. We found that the binding of KLF6 and PU.1 transcription factors occurred in the region -168/+69. Both perform a vital regulatory function in the transcription of bovine ELOVL6. Overexpression of ELOVL6 significantly upregulated the expression of peroxisome proliferator activated receptor γ (PPARγ), but inhibited the expression of fatty acid-binding protein 4 (FABP4), while silencing of ELOVL6 negatively regulated the messenger RNA expression level of PPARγ, FABP4, ACSL, and FATP1. In addition, ELOVL6 promotes adipocyte proliferation by regulating the cell-cycle genes' expression. Taken together, these findings provide useful information about the transcriptional regulation and functional mechanisms of bovine ELOVL6 in lipid metabolism and adipocyte proliferation in Qinchuan cattle.