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The role of miRNAs in mouse embryonic stem cells: A proteomics approach

Student thesis: Doctoral ThesisDoctor of Philosophy

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level. Studies in mouse embryonic stem cells (mESCs) where DGCR8; a major protein involved in their biogenesis has been knocked out, have shown that the global loss of canonical miRNAs results in cell cycle defects, delayed and reduced expression of markers of differentiation, and an inability to downregulate pluripotency markers upon differentiation. By conducting a 2D-DIGE study, comparing protein expression in wild type and DGCR8-/- mESCs, the aim was to study the effects that the loss of DGCR8 has on the proteome of mESCs when grown under proliferative conditions. The loss of DGCR8 in mESCs resulted in the deregulation of proteins with a chaperone function and those involved in glucose metabolism. Notably enzymes involved in glycolysis were reduced, whereas those involved in the TCA cycle were upregulated compared to wild type cells. mESCs are known to be highly glycolytic and the form of glucose metabolism used by cells has been linked to their capacity to differentiate. A second DIGE study was carried out on DGCR8-/- mESCs individually transfected with Embryonic Stem Cell Cycle specific (ESCC) miRNAs, to establish novel targets of these miRNAs and study their effect on the proteome. The study revealed that the ESCC miRNAs influence the expression of glucose metabolism proteins, notably Aldolase A, a key enzyme for glycolysis was identified in both studies as being an indirect target of the ESCC miRNAs. High resolution nuclear magnetic resonance spectroscopy further revealed differences in metabolism between the DGCR8-/- mESCs transfected with the ESCC miRNAs and those transfected with a control miRNA, indicative of a switch from predominantly glucose metabolism in the wild type mESCs to glutaminolysis for energy generation in the DGCR8-/-. Therefore the same miRNAs that control the embryonic stem cell cycle, also play a major role in the metabolic status of these cells, which may in turn play a role in the controlling the balance between pluripotency and differentiation. At the time of writing this is the first study using proteomic techniques to compare DGCR8-/- and wild type mESCs, and to explore the effect of the ESCC miRNAs.
Original languageEnglish
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Award date2018

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