RNA-Seq explores the functional role of the FGF10 gene in bovine adipocytes differentiation.

Objective: The present study was executed to explore the molecular mechanism of FGF10 gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their NCs in bovine adipocytes, and the MOI, transfection efficiency, interference efficiency were evaluated through qRT-PCR, western blotting and fluorescence microscopy. The lipid droplets, TG content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as C/EBPa, FABP4, PPARy, LPL, and FAS, similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPa, PPARy, FABP4, and LPL genes (P˂0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1774 DEGs (differentially expressed genes) including 157 up regulated and 1617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top KEGG pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.

Polymorphism of the PLIN1 gene and its association with body measures and ultrasound carcass traits in Qinchuan beef cattle.

The PLIN1 gene produces a phosphorylated protein wrapped in lipid droplets in adipocytes. This phosphorylation assists the mobilization of fat into adipose tissue. The purpose of the experiment was to study the polymorphism of the PLIN1 gene and its relationship with the body and carcass characteristics of Qinchuan cattle to find molecular genetic markers that can be used for breeding. The expression level of the PLIN1 gene was determined in various tissues by qRT-PCR. The results showed that the highest level of PLN1 expression was found in subcutaneous fat, followed by the heart and longissimus muscle, and the lowest level was found in the kidney. Five SNP loci of the PLIN1 gene were identified in 510 Qinchuan cattle, including g.3580T>C (SNP1), g.3898G>A (SNP2), g.8333G>A (SNP3), g.10517T>C (SNP4), and g.10538G>T (SNP5). The results show that SNP1, SNP2, SNP3, and SNP4 were moderately polymorphic (0.25 < PIC < 0.5), while SNP5 was minimally polymorphic (PIC < 0.25). SNP2, SNP3, and SNP5 were within Hardy-Weinberg equilibrium (P > 0.05), but SNP1 and SNP4 were not (P < 0.05). Correlation analysis showed that the five SNPs of the PLIN1 gene were correlated with back-fat depth, intramuscular fat, and chest depth of Qinchuan cattle. The double haplotype H2H4 in Qinchuan beef was associated with body and carcass traits. We conclude that variants mapped within PLIN1 can be used in marker-assisted selection for carcass quality and body traits in breed improvement programs for Qinchuan cattle.

The Role of MicroRNAs in Muscle Tissue Development in Beef Cattle.

In this review, we highlight information on microRNA (miRNA) identification and functional characterization in the beef for muscle and carcass composition traits, with an emphasis on Qinchuan beef cattle, and discuss the current challenges and future directions for the use of miRNA as a biomarker in cattle for breeding programs to improve meat quality and carcass traits. MicroRNAs are endogenous and non-coding RNA that have the function of making post-transcriptional modifications during the process of preadipocyte differentiation in mammals. Many studies claim that diverse miRNAs have an impact on adipogenesis. Furthermore, their target genes are associated with every phase of adipocyte differentiation. It has been confirmed that, during adipogenesis, several miRNAs are differentially expressed, including miR-204, miR-224, and miR-33. The development of mammalian skeletal muscle is sequentially controlled by somite commitment into progenitor cells, followed by their fusion and migration, the proliferation of myoblasts, and final modification into fast- and slow-twitch muscle fibers. It has been reported that miRNA in the bovine MEG3-DIO3 locus has a regulatory function for myoblast differentiation. Likewise, miR-224 has been associated with controlling the differentiation of bovine adipocytes by targeting lipoprotein lipase. Through the posttranscriptional downregulation of KLF6, miR-148a-3p disrupts the proliferation of bovine myoblasts and stimulates apoptosis while the miR-23a~27a~24-2 cluster represses adipogenesis. Additional to influences on muscle and fat, bta-mir-182, bta-mir-183, and bta-mir-338 represent regulators of proteolysis in muscle, which influences meat tenderness.

Function and characterization of the promoter region of perilipin 1 (PLIN1): Roles of E2F1, PLAG1, C/EBPß, and SMAD3 in bovine adipocytes.

Perilipin 1 (PLIN1) protein, also known as lipid droplet-associated protein, is encoded by the PLIN1 gene and is able to anchor itself to the membranes of lipid droplets. The phosphorylation of PLIN1 is critical for the mobilization of fat in adipose tissue and plays an important role in regulating lipolysis and lipid storage in adipocytes. However, research on the synthesis and lipid metabolism of lipid droplets by PLIN1 in bovine adipocytes is limited. In the present study, we found that bovine PLIN1 was highly expressed in subcutaneous adipose tissue. The highest level of PLIN1 mRNA expression in bovine adipocytes was observed on day 6 of differentiation. Moreover, the cytoplasmic subcellular localization of PLIN1 was observed in bovine preadipocytes. To elucidate the molecular mechanism of bovine PLIN1 transcriptional regulation, we cloned eight fragments containing the 5' regulatory region of the PLIN1 gene. The results showed that the -209/-17 bp region of the bovine PLIN1 gene was the core promoter region. Based on the transcriptional activities of the promoter vector fragments, the luciferase activity of the mutated fragment, the siRNA interference, and the results of the electrophoretic mobility shift assay (EMSA), we identified the binding sites of E2F transcription factor 1 (E2F1), pleiomorphic adenoma gene 1 (PLAG1), CCAAT enhancer binding protein beta (C/EBPß), and SMAD family member 3 (SMAD3) as the transcriptional activators or repressors of the core promoter region. Further experiments confirmed that the knockdown of the PLIN1 gene affected the ability of these transcription factors to regulate the lipid metabolism in bovine adipocytes. In conclusion, our results reveal a potential mechanism for the transcriptional regulation of PLIN1 in bovine adipocytes.