Cataloged from PDF version of thesis.Includes bibliographical reference (leaves 53-60).Thesis (M.S.): Bilkent University, Department of Molecular Biology and Genetics, İhsan Doğramacı Bilkent University, 2014.The proper functioning and the development of the cell is essential to the fitness of the
multicellular organisms - any significant disturbances in cellular mechanisms can lead to a
multitude of diseases or death. Among these conditions, the global rise in metabolic diseases
like obesity, diabetes and atherosclerosis draw significant research interest focus. Since the
prevalence of metabolic disorders in the developed and underdeveloped world is expected to
increase further in next decade; understanding the contributing cellular mechanisms is vital
for the development of new and effective diagnostic and therapeutic tools against this
devastating disease cluster.
Among the homeostatic cellular pathways important for health the Unfolded Protein Response
(UPR) is highly conserved from yeast to mammals. Aside from most conserved UPR branch
Inositol-requiring protein 1(IRE1), the mammalian UPR is composed of three different
pathways regulated by IRE1, eukaryotic translation initiation factor 2-alpha kinase 3 (PERK),
and activating transcription factor 6 (ATF6). The UPR signaling is activated in response to the
accumulation of unfolded or misfolded proteins in ER that leads to endoplasmic reticulum
(ER) stress. The goal of the UPR is to re-establish ER homeostasis via inhibition of further protein translation and promoting protein folding. In the case of severe or unresolved ER
stress, UPR instead triggers a programmed cell death.
Recent studies indicate that noncoding regulatory RNAs such as microRNAs (miRNAs) play
important role in both upstream and downstream of the UPR. In this thesis, the regulation of
miRNA expression by the different UPR arms are examined in macrophages under lipidinduced
or lipotoxic ER stress conditions. The results of PCR array studies of RNA obtained
from mouse macrophages stressed with a saturated fatty acid, palmitate (PA) , revealed
multiple differentially regulated miRNAs. Among these miRNAs, significantly regulated ones
were further examined for their regulation by the different arms of the UPR. Towards this end
several complementary approaches were taken: First, significantly regulated microRNAs
from microRNA PCR array results were analyzed. Next, macrophages were treated with
palmitate after transfection with IRE1 and PERK silencer RNA (siRNA) to assess the role of
UPR arms in lipid regulated miRNA regulation and the expression of relevant miRNAs was
examined in treated macrophages. As an alternative method, macrophages were treated
simultaneously with palmitate and specific inhibitors for IRE1’s endoribonuclease or PERK’s
kinase activity. Then miRNA expressions were further examined in IRE1 knock-out mouse
embryonic fibroblast (MEF) cell lines transfected with the wild type (WT) IRE or the
endoribonuclease domain inactive (RD) mutant of IRE1 to verify the specific regulation of the
miRNA by the IRE1’s endoribonuclease activity. As a result, upregulation of miR-2137
expression by palmitate was determined as IRE1-endoribonuclease dependent. Next, potential
target mRNAs were examined by the overexpression or knock-down of miR-2137 in
macrophages. One possible target mRNA was found to be inositol polyphosphate
phosphatase-like 1 (Innpl1) . Aside from miR-2137, miR-33 also showed significant alteration
upon PA treatment in macrophages. Since the role of miR-33 in atherosclerosis, obesity and insulin resistance is well established, its expression was studied further in RAW 264.7
macrophage cell line and bone marrow-derived primary macrophages after IRE1 and PERK
knock-down with siRNA. ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1),
a known target of miR-33, was investigated as down-stream target of miR-33 in PA treated
macrophages, in an IRE1 dependent manner.
The results of this study uncovered new UPR regulated miRNAs under lipid stress in
macrophages. Excess lipid is one of the prominent causes in metabolic diseases – obesity,
atherosclerosis, insulin resistance – and these UPR regulated miRNAs may explain the
underlying mechanism behind this set of diseases. Furthermore, the possible gene targets for
these miRNAs could be responsible for progression of such conditions. Further studies are
needed to reveal the exact mechanisms that can lead to the development of novel therapeutic
approaches.by Erdem Murat Terzi.M.S
