ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis.

Acyl-coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid ß-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at -1142 to -1129 bp, -831 to -826 bp, and -303 to -298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3'UTR (3'UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid ß-oxidation.

Characterization of XR_311113.2 as a MicroRNA Sponge for Pre-ovulatory Ovarian Follicles of Goats via Long Noncoding RNA Profile and Bioinformatics Analysis.

Long noncoding RNAs (lncRNAs) were identified recently as a large class of noncoding RNAs (ncRNAs) with a length ≥200 base pairs (bp). The function and mechanism of lncRNAs have been reported in a growing number of species and tissues. In contrast, the regulatory mechanism of lncRNAs in the goat reproductive system has rarely been reported. In the present study, we sequenced and analyzed the lncRNAs using bioinformatics to identify their expression profiles. As a result, 895 lncRNAs were predicted in the pre-ovulatory ovarian follicles of goats. Eighty-eight lncRNAs were differentially expressed in the Macheng black goat when compared with Boer goat. In addition, the lncRNA XR_311113.2 acted as a sponge of chi-miR-424-5p, as assessed via a luciferase activity assay. Taken together, our findings demonstrate that lncRNAs have potential effects in the ovarian follicles of goats and may represent a promising new research field to understand follicular development.

Skin transcriptome profiles associated with black- and white-coated regions in Boer and Macheng black crossbred goats.

To increase the current understanding of the gene-expression profiles in different skin regions associated with different coat colors and identify key genes for the regulation of color patterns in goats, we used the Illumina RNA-Seq method to compare the skin transcriptomes of the black- and white-coated regions containing hair follicles from the Boer and Macheng Black crossbred goat, which has a black head and a white body. Six cDNA libraries derived from skin samples of the white-coated region (n = 3) and black-coated region (n = 3) were constructed from three full-sib goats. On average, we obtained approximately 76.5 and 73.5 million reads for skin samples from black- and white-coated regions, respectively, of which 75.39% and 76.05% were covered in the genome database. A total of 165 differentially expressed genes (DEGs) were detected between these two color regions, among which 110 were upregulated and 55 were downregulated in the skin samples of white- vs. black-coated regions. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that some of these DEGs may play an important role in controlling the pigmentation of skin or hair follicles. We identified three key DEGs, i.e., Agouti, DCT, and TYRP1, in the pathway related to melanogenesis in the different skin regions of the crossbred goat. DCT and TYRP1 were downregulated and Agouti was upregulated in the skin of the white-coated region, suggesting a lack of mature melanocytes in this region and that Agouti might play a key developmental role in color-pattern formation. All data sets (Gene Expression Omnibus) are available via public repositories. In addition, MC1R was genotyped in 200 crossbred goats with a black head and neck. Loss-of-function mutations in MC1R as well as homozygosity for the mutant alleles were widely found in this population. The MC1R gene did not seem to play a major role in determining the black head and neck in our crossbred goats. Our study provides insights into the transcriptional regulation of two distinct coat colors, which might serve as a key resource for understanding coat color pigmentation in goats. The region-specific expression of Agouti may be associated with the distribution of pigments across the body in Boer and Macheng Black crossbred goats.

Circular RNA profiling reveals chi_circ_0008219 function as microRNA sponges in pre-ovulatory ovarian follicles of goats (Capra hircus).

Circular RNAs (circRNAs) are a new class of non-coding RNAs in animals and are a novel target of non-coding RNA (ncRNA) regulation. The mechanism and function of circRNAs have been reported in some species and tissues. However, there is little available information on the functions of circRNAs in the goat reproductive system. In the present study, we deeply sequenced and analyzed circRNAs through bioinformatics to reveal the expression profiles, and predicted 13,950 circRNAs in the pre-ovulatory ovarian follicles of goats for the first time. Thirty-seven circRNAs were differentially expressed in the Boer goat compared with the Macheng black goat. The chi_circ_0008219 was involved in a vast circRNA-miRNA-mRNA co-expression network. Via a luciferase activity assay, chi_circ_0008219 is observed to sponge to 3 ovarian follicle-related miRNAs. These findings demonstrate that circRNAs have potential effects in the ovarian follicles of ewes and may represent a promising new research field in ovarian follicular development.

Association analysis of HSP70A1A haplotypes with heat tolerance in Chinese Holstein cattle.

Single-nucleotide polymorphisms (SNPs) in the coding and untranslated regions of heat shock 70 kDa protein 1A (HSP70A1A), an inducible molecular chaperone that is responsible for cellular protection against heat stress, have been reported as being associated with heat tolerance. A fragment of the HSP70A1A gene was amplified in Chinese Holstein cattle and eight novel mutations were found. We performed comprehensive linkage disequilibrium (LD) and haplotype analyses of the eight SNPs of the HSP70A1A gene and examined their involvement in heat resistance in 600 Chinese Holstein cattle. Our results revealed the presence of significant differences between individuals carrying haplotype 1 and those without haplotype 1 for most of the heat-tolerance traits. Haplotype 1 increased the risk of heat stress; however, association analysis of its combination with haplotype 2 showed the lowest rectal temperature and red blood cell K(+) level, moderate respiratory rate, and the highest red blood cell NKA level, suggesting a heterozygote advantage in the penetration of the phenotype. Protein expression levels in white blood cells among haplotype combinations further confirmed the hypothesis that heterozygotes for haplotypes 1 and 2 are more sensitive to heat stress. We presume that these mutations may be useful in the future as molecular genetic markers to assist selection for heat tolerance in cattle.

Characterization of porcine SKIP gene in skeletal muscle development: polymorphisms, association analysis, expression and regulation of cell growth in C2C12 cells.

Skeletal muscle and kidney-enriched inositol phosphatase (SKIP) was identified as a 5'-inositol phosphatase that hydrolyzes phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) to PI(3,4)P2 and negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling in skeletal muscle. In this study, two new single nucleotide polymorphisms (SNPs) in porcine SKIP introns 1 and 6 were detected. The C1092T locus in intron 1 showed significant associations with some meat traits, whereas the A17G locus in intron 6 showed significant associations with some carcass traits. Expression analysis showed that porcine SKIP is upregulated at d 65 of gestation and Meishan fetuses have higher and prolonged expression of SKIP compared to Large White at d 100 of gestation. Ectopic expression of porcine SKIP decreased insulin-induced cell proliferation and promoted serum starvation-induced cell cycle arrest in G0/G1 phase in C2C12. Our results suggest that SKIP plays a negative regulatory role in skeletal muscle development partly by preventing cell proliferation.

Molecular characterization, expression pattern, and association analysis with carcass traits of the porcine SHIP2 gene.

Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) has been identified as 5'-inositol phosphatase that hydrolyzes PI(3,4,5)P(3) to PI(3,4)P(2), which negatively regulates insulin-induced Akt signaling in skeletal muscle. In this study, we obtained a 3,795-bp mRNA sequence of porcine SHIP2 that included the full coding region for a protein of 1,264 amino acids. With the use of comparative mapping, we mapped this gene to SSC9 p23-24, where many QTLs affect average backfat thickness, average daily weight gain (birth-10 weeks), adipocyte number, belly fat area, and mid-back fat traits. As a candidate gene for carcass traits, a novel single nucleotide polymorphism in intron 21 (A > G) was detected by PCR-RFLP. The results showed that the AA genotype had higher skin percentage, shoulder fat thickness, and m. longissimus dorsi width, but lower m. longissimus dorsi height compared with the genotype GG (P < 0.05), and that allele G appeared to be associated with an increase in the growth trait. SHIP2 was expressed abundantly in skeletal muscle tissue and was transcriptionally decreased during the proliferative phase, but increased in the intermediate stages of muscle differentiation. Analysis of the porcine SHIP2 promoter sequence demonstrated that the E2F element is involved in downregulating SHIP2 mRNA expression in proliferating myoblasts. Using RNAi, we found that the MyoD transcription factor played a role in upregulating SHIP2 expression in differentiating myotubes. In summary, we suggest that SHIP2 might play a role in the regulation of skeletal muscle development in pigs.