MicroRNAs in bovine adipose tissue: genomic context, expression and function
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ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs found to regulate several biological processes including adipogenesis. Understanding adipose tissue regulation is critical for beef cattle as fat is an important determinant of beef quality and nutrient value . This study analyzed the association between genomic context characteristics of miRNAs with their expression and function in bovine adipose tissue. Twenty-four subcutaneous adipose tissue biopsies were obtained from eight British-continental crossbred steers at 3 different time points . Total RNA was extracted and miRNAs were profiled using a miRNA microarray with expression further validated by qRT-PCR. A total of 224 miRNAs were detected of which 155 were expressed in all steers (n=8), and defined as the core miRNAs of bovine subcutaneous adipose tissue. Core adipose miRNAs varied in terms of genomic location (59.5% intergenic, 38.7% intronic, 1.2% exonic, and 0.6% mirtron), organization (55.5% non-clustered and 44.5% clustered), and conservation (49% highly conserved, 14% conserved and 37% poorly conserved). Clustered miRNAs and highly conserved miRNAs were more highly expressed (p<0.05) and had more predicted targets than non-clustered or less conserved miRNAs (p<0.001). A total of 34 miRNAs were coordinately expressed, being part of six identified relevant networks. Two intronic miRNAs (miR-33a and miR-1281) were shown to have coordinated expression with their host genes which are involved in lipid metabolism, suggesting these miRNAs may also play a role in regulation of lipid metabolism/adipogenesis of bovine adipose tissue. Furthermore, a total of 17 bovine specific miRNAs were predicted to be involved in the regulation of energy balance in adipose tissue. These findings improve our understanding on the behavior of miRNAs in the regulation of bovine adipogenesis and fat metabolism as it reveals that miRNA expression patterns and functions are associated with miRNA genomic organization and conservation in bovine adipose tissue.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs found to regulate several biological processes including adipogenesis. Understanding adipose tissue regulation is critical for beef cattle as fat is an important determinant of beef quality and nutrient value . This study analyzed the association between genomic context characteristics of miRNAs with their expression and function in bovine adipose tissue. Twenty-four subcutaneous adipose tissue biopsies were obtained from eight British-continental crossbred steers at 3 different time points . Total RNA was extracted and miRNAs were profiled using a miRNA microarray with expression further validated by qRT-PCR. A total of 224 miRNAs were detected of which 155 were expressed in all steers (n=8), and defined as the core miRNAs of bovine subcutaneous adipose tissue. Core adipose miRNAs varied in terms of genomic location (59.5% intergenic, 38.7% intronic, 1.2% exonic, and 0.6% mirtron), organization (55.5% non-clustered and 44.5% clustered), and conservation (49% highly conserved, 14% conserved and 37% poorly conserved). Clustered miRNAs and highly conserved miRNAs were more highly expressed (p<0.05) and had more predicted targets than non-clustered or less conserved miRNAs (p<0.001). A total of 34 miRNAs were coordinately expressed, being part of six identified relevant networks. Two intronic miRNAs (miR-33a and miR-1281) were shown to have coordinated expression with their host genes which are involved in lipid metabolism, suggesting these miRNAs may also play a role in regulation of lipid metabolism/adipogenesis of bovine adipose tissue. Furthermore, a total of 17 bovine specific miRNAs were predicted to be involved in the regulation of energy balance in adipose tissue. These findings improve our understanding on the behavior of miRNAs in the regulation of bovine adipogenesis and fat metabolism as it reveals that miRNA expression patterns and functions are associated with miRNA genomic organization and conservation in bovine adipose tissue. In this study, a total of 24 subcutaneous adipose tissue samples were analyzed by microRNA microarrays. The samples were derived from eight steers at three different ages (12, 13.5 and 15 months).
Project description:MicroRNAs are a class of molecular regulators found to participate in numerous biological processes, including adipogenesis. However, whether dietary changes impact on microRNA (miRNA) in ruminants has not been reported. Therefore, this study aimed to evaluate the dietary effect on miRNA expression in subcutaneous (backfat) and visceral fat depots (perirenal fat) from beef steers fed with different diets containing high or low fat levels. Fat tissues were collected from 16 Hereford x Aberdeen Angus cross bred steers (15.5 month old) fed high fat diet (5.85% fat, n=8) or control diet (1.95% fat, n=8). Total RNA from each animal was subjected to miRNA microarray analysis using a customized Agilent miRNA microarray containing 672 bovine miRNA probes. Expression of miRNAs was not equally detected under two diets; 169 miRNAs were commonly expressed while 75 were diet specific. The number of miRNAs detected per animal under high fat diet was higher than those fed control diet (p= 0.037 in subcutaneous fat and p= 0.002 in visceral fat).. Further qRT-PCR analysis confirmed that the expression of some miRNAs was highly influenced by diet (miR-19a, -92a, -92b, -101, -103, -106, -142-5p, and 296) or fat depot (miR-196a and -2454). Our results revealed that the miRNA expression can be influenced by types of fat tissues or diet, suggesting that miRNAs may regulate bovine adipogenesis when diet alters. In this study, a total of 32 adipose tissue samples were analyzed by microRNA microarrays, being 16 subcutaneus (backfat) and 16 visceral (perirenal fat) fat depots were collected from 16 animals (Control diet = 8) (High fat diet = 8).
Project description:MicroRNAs are a class of molecular regulators found to participate in numerous biological processes, including adipogenesis. However, whether dietary changes impact on microRNA (miRNA) in ruminants has not been reported. Therefore, this study aimed to evaluate the dietary effect on miRNA expression in subcutaneous (backfat) and visceral fat depots (perirenal fat) from beef steers fed with different diets containing high or low fat levels. Fat tissues were collected from 16 Hereford x Aberdeen Angus cross bred steers (15.5 month old) fed high fat diet (5.85% fat, n=8) or control diet (1.95% fat, n=8). Total RNA from each animal was subjected to miRNA microarray analysis using a customized Agilent miRNA microarray containing 672 bovine miRNA probes. Expression of miRNAs was not equally detected under two diets; 169 miRNAs were commonly expressed while 75 were diet specific. The number of miRNAs detected per animal under high fat diet was higher than those fed control diet (p= 0.037 in subcutaneous fat and p= 0.002 in visceral fat).. Further qRT-PCR analysis confirmed that the expression of some miRNAs was highly influenced by diet (miR-19a, -92a, -92b, -101, -103, -106, -142-5p, and 296) or fat depot (miR-196a and -2454). Our results revealed that the miRNA expression can be influenced by types of fat tissues or diet, suggesting that miRNAs may regulate bovine adipogenesis when diet alters.
Project description:The posttranscriptional gene regulation mediated by microRNA (miRNA) plays an important role in various species. Recently, a large number of miRNAs and their expression patterns have been identified. However, to date, limited miRNAs have been reported to modulate adipogenesis and lipid deposition in beef cattle. Total RNAs from Chinese Qinchuan bovine backfat at fetal and adult stages were used to construct small RNA libraries for Illumina next-generation sequencing. A total of 13,915,411 clean reads were obtained from a fetal library and 14,244,946 clean reads from an adult library. In total, 475 known and 36 novel miRNA candidates from backfat were identified. The nucleotide bias, base editing, and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 15 specific miRNAs were detected, and the results showed that bta-miRNAn25 and miRNAn26 were highly expressed in backfat tissue, suggesting these small RNAs play a role in the development and maintenance of bovine subcutaneous fat tissue. Putative targets for miRNAn25 and miRNAn26 were predicted, and the 61 most significant target transcripts were related to lipid and fatty acid metabolism. Of interest, the canonical pathway and gene networks analyses revealed that PPARα/RXRα activation and LXR/RXR activation were important components of the gene interaction hierarchy results. In the present study, we explored the backfat miRNAome differences between cattle of different developmental stages, expanding the expression repertoire of bovine miRNAs that could contribute to further studies on the fat development of cattle. The expression analysis of the differential target genes of miRNAn25 and miRNAn26 showed potential gene networks that affect lipid and fatty acid metabolism. These results may help in the design of new intervention strategies to improve beef quality. Examination of Chinese Qinchuan bovine backfat miRNAs by deep sequencing.
Project description:The posttranscriptional gene regulation mediated by microRNA (miRNA) plays an important role in various species. Recently, a large number of miRNAs and their expression patterns have been identified. However, to date, limited miRNAs have been reported to modulate adipogenesis and lipid deposition in beef cattle. Total RNAs from Chinese Qinchuan bovine backfat at fetal and adult stages were used to construct small RNA libraries for Illumina next-generation sequencing. A total of 13,915,411 clean reads were obtained from a fetal library and 14,244,946 clean reads from an adult library. In total, 475 known and 36 novel miRNA candidates from backfat were identified. The nucleotide bias, base editing, and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 15 specific miRNAs were detected, and the results showed that bta-miRNAn25 and miRNAn26 were highly expressed in backfat tissue, suggesting these small RNAs play a role in the development and maintenance of bovine subcutaneous fat tissue. Putative targets for miRNAn25 and miRNAn26 were predicted, and the 61 most significant target transcripts were related to lipid and fatty acid metabolism. Of interest, the canonical pathway and gene networks analyses revealed that PPARα/RXRα activation and LXR/RXR activation were important components of the gene interaction hierarchy results. In the present study, we explored the backfat miRNAome differences between cattle of different developmental stages, expanding the expression repertoire of bovine miRNAs that could contribute to further studies on the fat development of cattle. The expression analysis of the differential target genes of miRNAn25 and miRNAn26 showed potential gene networks that affect lipid and fatty acid metabolism. These results may help in the design of new intervention strategies to improve beef quality.
Project description:Neotyphodium coenophialum is an endophytic fungus that infects most tall fescue (Festuca arundinacea) pastures that are commonly used in animal grazing systems in the United States. Beef cattle grazing such pastures are impaired in health and production performance, resulting in a large economic loss in US food-animal production systems. Based on the clinical symptoms and laboratory analyses of blood, it was hypothesized that such affected cattle display liver-specific changes in the expression of gene transcripts that are associated with the metabolic enzymes and transporters critical for beef health and performance. Microarray analysis using the GeneChip Bovine Genome Array (Affymetrix, Inc., Santa Clara, CA) was conducted to determine if grazing endophyte-infected tall fescue pastures affects the liver gene expression profiles of growing beef steers. Nineteen steers were assigned to graze either a low toxic endophyte tall fescue-mixed grass (LE treatment, 5.7 ha, n = 9) or a high toxic endophyte infected tall fescue (HE treatment, 5.7 ha, n = 10) pasture located in the University of Kentucky Agricultural Research Center. All steers had ad libitum access to fresh water and an industry standard mineral-vitamin supplement. 88 days grazing on pasture. Approximately 2 g of tissue from the right lobe of the liver of each steer were collected for RNA extraction and microarray analysis.
Project description:Adipose tissue abundance relies partly on the factors that regulate adipogenesis, i.e. proliferation and differentiation of adipocytes. While the transcriptional program that initiates adipogenesis is well-known, the importance of microRNAs in adipogenesis is less well studied. We thus set out to investigate whether miRNAs would be actively modulated during adipogenesis and obesity. Several models exist to study adipogenesis in vitro, of which the cell line 3T3-L1 is probably the most well known, albeit not the most physiologically appropriate. We used a microarray strategy to provide a global profile of miRNAs in brown and white primary murine adipocytes (prior to and following differentiation) and evaluated the similarity of the responses to non-primary cell models, through literature data-mining. We found 65 miRNAs regulated during in vitro adipogenesis in primary adipocytes. When we compared our primary adipocyte profiles with those of cell lines reported in the literature, we found a high degree of difference in adipogenesis-regulated miRNAs. We evaluated the expression of 10 of our adipogenesis-regulated miRNAs using real-time qPCR and then selected 5 miRNAs that showed robust expression levels and profiled these by qPCR in subcutaneous adipose tissue of 20 humans with a range of body mass indices (BMI, range=21-48). Of the miRNAs tested, mir-21 was both highly expressed in human adipose tissue and positively correlated with BMI (R2=0.49, p<0.001). In conclusion, we provide the preliminary analysis of miRNAs important for primary cell in vitro adipogenesis and find that the inflammation-associated miRNA, mir-21, is up-regulated in subcutaneous adipose tissue in human obesity. 3 samples of pre adipocytes isolated from brown adipose tissue examined pre and post differentiation to brown adipocytes. 3 samples of pre-adipocytes isolated from white adipose tissue and examined pre and post differentiation to adipocytes.
Project description:Beef marbling is caused by intramuscular deposition, and it is an economically important trait in the beef industry. Vitamin A (VA) is an important feed supplement for cattle, but it can hinder marbling if provided in excess. In cattle, VA forms various derivatives such as all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA). Therefore, we investigated the genes involved in bovine intramuscular adipogenesis after VA treatment with ATRA and 9cRA. Differential gene expression levels were validated by microarray analysis in a clonal bovine intramuscular preadipocyte (BIP) cell line derived from the intramuscular adipose tissue of Japanese Black cattle. BIP cells were harvested six days after adipogenic stimulation with either 1 μM ATRA, 1 μM 9cRA, or nonretinoic acids control. The ATRA- and 9cRA-treated cells exhibited reduced transcription of genes involved in the circulatory system and muscle development compared with the no retinoic acid (RA) treatment. In addition, the ATRA- and 9cRA-treated cells exhibited increased transcription of genes involved in the immune system, protein kinase B signaling, and responses to various stimuli. These results demonstrate the lower expression of muscle development in ATRA- and 9cRA-treated BIP cells during adipogenesis.
Project description:Adipose tissue abundance relies partly on the factors that regulate adipogenesis, i.e. proliferation and differentiation of adipocytes. While the transcriptional program that initiates adipogenesis is well-known, the importance of microRNAs in adipogenesis is less well studied. We thus set out to investigate whether miRNAs would be actively modulated during adipogenesis and obesity. Several models exist to study adipogenesis in vitro, of which the cell line 3T3-L1 is probably the most well known, albeit not the most physiologically appropriate. We used a microarray strategy to provide a global profile of miRNAs in brown and white primary murine adipocytes (prior to and following differentiation) and evaluated the similarity of the responses to non-primary cell models, through literature data-mining. We found 65 miRNAs regulated during in vitro adipogenesis in primary adipocytes. When we compared our primary adipocyte profiles with those of cell lines reported in the literature, we found a high degree of difference in adipogenesis-regulated miRNAs. We evaluated the expression of 10 of our adipogenesis-regulated miRNAs using real-time qPCR and then selected 5 miRNAs that showed robust expression levels and profiled these by qPCR in subcutaneous adipose tissue of 20 humans with a range of body mass indices (BMI, range=21-48). Of the miRNAs tested, mir-21 was both highly expressed in human adipose tissue and positively correlated with BMI (R2=0.49, p<0.001). In conclusion, we provide the preliminary analysis of miRNAs important for primary cell in vitro adipogenesis and find that the inflammation-associated miRNA, mir-21, is up-regulated in subcutaneous adipose tissue in human obesity. A global transcriptomic survey of subcutaneous adipose tissue from human subjects characterised as having normal glucose tolerance, glucose intolerance or frank type 2 diabetes.
Project description:Growing and finishing phases are two important animal production stages, which differ fundamentally in compositional growth. However, the physiological mechanisms altered concomitantly with the shift in whole-body compositional gain as cattle fatten (growing vs. finished steers), are poorly understood. Microarray analysis using the Bovine Gene 1.0 ST Array was conducted to determine shifts in hepatic genomic expression profiles of growing vs. finishing beef steers. The specific overall hypothesis tested was that genes involved in amino acid, carbohydrate and lipid metabolism, antioxidant capacity and immune responses were differentially expressed in growing vs. finishing steers.