• Alterations in hepatic miRNA expression during negative energy balance in postpartum dairy cattle

      Fatima, Attia; Waters, Sinead M.; O'Boyle, Padraig; Seoighe, Cathal; Morris, Dermot G (Biomed Central, 2014-01-15)
      Abstract Background Negative energy balance (NEB), an altered metabolic state, occurs in early postpartum dairy cattle when energy demands to support lactation exceed energy intake. During NEB the liver undergoes oxidative stress and increased breakdown of fatty acids accompanied by changes in gene expression. It is now known that micro RNAs (miRNA) can have a role in mediating such alterations in gene expression through repression or degradation of target mRNAs. miRNA expression is known to be altered by metabolism and environmental factors and miRNAs are implicated in expression modulation of metabolism related genes. Results miRNA expression was profiled in the liver of moderate yielding dairy cattle under severe NEB (SNEB) and mild NEB (MNEB) using the Affymetrix Gene Chip miRNA_2.0 array with 679 probe sets for Bos-taurus miRNAs. Ten miRNAs were found to be differentially expressed using the ‘samr’ statistical package (delta = 0.6) at a q-value FDR of < 12%. Five miRNAs including miR-17-5p, miR-31, miR-140, miR-1281 and miR-2885 were validated using RT-qPCR, to be up-regulated under SNEB. Liver diseases associated with these miRNAs include non-alcoholic fatty liver (NAFLD) and hepatocellular carcinoma (HCC). miR-140 and miR-17-5p are known to show differential expression under oxidative stress. A total of 32 down-regulated putative target genes were also identified among 418 differentially expressed hepatic genes previously reported for the same animal model. Among these, GPR37 (G protein-coupled receptor 37), HEYL (hairy/enhancer-of-split related with YRPW motif-like), DNJA1, CD14 (Cluster of differentiation 14) and GNS (glucosamine (N-acetyl)-6-sulfatase) are known to be associated with hepatic metabolic disorders. In addition miR-140 and miR-2885 have binding sites on the most down-regulated of these genes, FADS2 (Fatty acid desaturase 2) which encodes an enzyme critical in lipid biosynthesis. Furthermore, HNF3-gamma (Hepatocyte nuclear factor 3-gamma), a hepatic transcription factor (TF) that is involved in IGF-1 expression regulation and maintenance of glucose homeostasis is a putative target of miR-31. Conclusions This study shows that SNEB affects liver miRNA expression and these miRNAs have putative targets in hepatic genes down-regulated under this condition. This study highlights the potential role of miRNAs in transcription regulation of hepatic gene expression during SNEB in dairy cattle. Background Negative energy balance (NEB), an altered metabolic state, occurs in early postpartum dairy cattle when energy demands to support lactation exceed energy intake. During NEB the liver undergoes oxidative stress and increased breakdown of fatty acids accompanied by changes in gene expression. It is now known that micro RNAs (miRNA) can have a role in mediating such alterations in gene expression through repression or degradation of target mRNAs. miRNA expression is known to be altered by metabolism and environmental factors and miRNAs are implicated in expression modulation of metabolism related genes. Results miRNA expression was profiled in the liver of moderate yielding dairy cattle under severe NEB (SNEB) and mild NEB (MNEB) using the Affymetrix Gene Chip miRNA_2.0 array with 679 probe sets for Bos-taurus miRNAs. Ten miRNAs were found to be differentially expressed using the ‘samr’ statistical package (delta = 0.6) at a q-value FDR of < 12%. Five miRNAs including miR-17-5p, miR-31, miR-140, miR-1281 and miR-2885 were validated using RT-qPCR, to be up-regulated under SNEB. Liver diseases associated with these miRNAs include non-alcoholic fatty liver (NAFLD) and hepatocellular carcinoma (HCC). miR-140 and miR-17-5p are known to show differential expression under oxidative stress. A total of 32 down-regulated putative target genes were also identified among 418 differentially expressed hepatic genes previously reported for the same animal model. Among these, GPR37 (G protein-coupled receptor 37), HEYL (hairy/enhancer-of-split related with YRPW motif-like), DNJA1, CD14 (Cluster of differentiation 14) and GNS (glucosamine (N-acetyl)-6-sulfatase) are known to be associated with hepatic metabolic disorders. In addition miR-140 and miR-2885 have binding sites on the most down-regulated of these genes, FADS2 (Fatty acid desaturase 2) which encodes an enzyme critical in lipid biosynthesis. Furthermore, HNF3-gamma (Hepatocyte nuclear factor 3-gamma), a hepatic transcription factor (TF) that is involved in IGF-1 expression regulation and maintenance of glucose homeostasis is a putative target of miR-31. Conclusions This study shows that SNEB affects liver miRNA expression and these miRNAs have putative targets in hepatic genes down-regulated under this condition. This study highlights the potential role of miRNAs in transcription regulation of hepatic gene expression during SNEB in dairy cattle.
    • The miRNAome of the postpartum dairy cow liver in negative energy balance

      Fatima, Attia; Lynn, David J; O'Boyle, Padraic; Seoighe, Cathal; Morris, Dermot (Biomed Central, 2014-04-12)
      Background: Negative energy balance (NEB) is an altered metabolic state in high yielding cows that occurs during the first few weeks postpartum when energy demands for lactation and maintenance exceed the energy supply from dietary intake. NEB can, in turn, lead to metabolic disorders and to reduced fertility. Alterations in the expression of more than 700 hepatic genes have previously been reported in a study of NEB in postpartum dairy cows. miRNAs (microRNA) are known to mediate many alterations in gene expression post transcriptionally. To study the hepatic miRNA content of postpartum dairy cows, including their overall abundance and differential expression, in mild NEB (MNEB) and severe NEB (SNEB), short read RNA sequencing was carried out. To identify putative targets of differentially expressed miRNAs among differentially expressed hepatic genes reported previously in dairy cows in SNEB computational target identification was employed. Results: Our results indicate that the dairy cow liver expresses 53 miRNAs at a lower threshold of 10 reads per million. Of these, 10 miRNAs accounted for greater than 95% of the miRNAome (miRNA content). Of the highly expressed miRNAs, miR-122 constitutes 75% followed by miR-192 and miR-3596. Five out of thirteen let-7 miRNA family members are also among the highly expressed miRNAs. miR-143, down-regulated in SNEB, was found to have 4 putative up-regulated gene targets associated with SNEB including LRP2 (low density lipoprotein receptor-related protein 2), involved in lipid metabolism and up-regulated in SNEB. Conclusions: This is the first liver miRNA-seq profiling study of moderate yielding dairy cows in the early postpartum period. Tissue specific miR-122 and liver enriched miR-192 are two of the most abundant miRNAs in the postpartum dairy cow liver. miR-143 is significantly down-regulated in SNEB and putative targets of miRNA-143 which are up-regulated in SNEB, include a gene involved in lipid metabolism.