Analysis of hepatic microRNA expression in postpartum dairy cows in negative energy balance
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This thesis sets about to answer three research questions grounded in transcriptional regulation of hepatic genes in the high yielding postpartum dairy cow under negative energy balance (NEB). For this purpose we used liver tissue from a previously developed dairy cow model comprising of cows under mild negative energy balance (MNEB) and severe negative energy balance (SNEB). The first research question relates to what is the overall expression profile distribution of hepatic miRNAs in the early postpartum period under both conditions and whether hepatic miRNA expression is altered during SNEB. The second question is an in silico identification of the gene targets of these miRNAs among sets of differentially expressed hepatic genes previously reported in the same animals. The third question is to determine the functional role of miRNAs in regulating putative target mRNAs. Two high-throughput techniques were used to determine hepatic miRNA expression under NEB in a dairy cattle model. First microarray expression profiling was done to determine the differential expression of hepatic miRNAs under SNEB. Five miRNAs were differentially expressed including miR-140, 31, 17-5p, 1281 and 2885. For all differentially expressed miRNAs, in-slico target prediction was carried out on a set of 418 differentially expressed liver genes previously reported in the same animals. A custom Perl script from TargetScan was used to identify putative target sites on bovine 3'UTRs retrieved from Ensemble. Overall 32 down-regulated target genes were identified, including those relevant to NEB such as genes associated with lipid and glucose metabolism and homeostasis and the somatotropic axis. miR-2885 has binding sites in the 3'UTR of FADS2, a lipogenic gene which is strongly down-regulated under SNEB. In addition, an up-regulated hepatic transcription factor, HNF3-gamma involved in IGF-1 expression is a putative target of miR-31. In the second study the overall abundance and distribution of miRNAs under both MNEB and SNEB was profiled using next generation sequencing. Overall, liverspecific miR-122 constituted 75% of expressed miRNA. SNEB resulted in the down-regulation of miR-143, which has been associated with fatty acid metabolism.Overall 16 up-regulated putative target genes of miR-143 were identified in a set of differentially expressed hepatic genes of same animals from micro-array and RNA-seq based previous NEB model studies. Among these SLC2A12 and LRP2 have roles in glucose and lipid metabolism.The third study aimed to determine the functional role of miR-2885 in the regulation of FADS2 expression. The inhibition of miR-2885 was carried out in an MDBK (Madin Darby Kidney) cell line through lipid-based transfection of a miR-2885 inhibitor and its effect on the expression of FADS2 was determined. FADS2 expression was up-regulated over 5-fold validating a role for miR-2885 in FADS2 regulation.
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