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Title: Regulatory Role of miR-93 in Autophagy Induction
Authors: LU KAIHUI
Keywords: microRNA, autophagy, Ulk1, miR-93, 3'UTR, STAT3
Issue Date: 11-Jan-2012
Citation: LU KAIHUI (2012-01-11). Regulatory Role of miR-93 in Autophagy Induction. ScholarBank@NUS Repository.
Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression. miRNAs play multiple functions in many essential cellular processes such as proliferation, apoptosis and differentiation. Autophagy is a conserved cellular process in which cell digests spent components and organelles to regenerate energy and ?building blocks? to cope with various kinds of stress. To date, the effect of autophagy on miRNA expression and the corresponding mechanisms through which miRNAs regulate autophagy are still not clear. Therefore, the main objective of this study is to investigate the effect of autophagy induction on endogenous miRNA expression and the corresponding mechanisms through which miRNAs regulate autophagy. Here, we first screened the differential expression of miRNAs by microarray when autophagy was induced by starvation in Atg5 wide type mouse embryonic fibroblasts (WT MEF) and Atg5 knock out mouse embryonic fibroblasts (KO MEF). Starvation and Rapamycin treatments are established common inducers for autophagy. The MEF cells were chosen as they were among the most popular cell line models for the current autophagy research, knock out of Atg5 can efficiently block autophagy induced by starvation or Rapamycin. Among these miRNAs, miR-93 and miR-181a were up-regulated while miR-221 was down-regulated when autophagy was induced. Such changes were confirmed using real time qRT-PCR in cells undergoing autophagy induced by either starvation or Rapamycin. Those miRNAs deregulated in the period of autophagy induction were predicted to target a variety of autophagy related genes, indicating the important regulatory role of miRNAs in autophagy. miR-93 was selected for further examination. Through screening of the predicted autophagy related target genes, we successfully identified Ulk1 as a novel target gene of miR-93, based on the following evidence: (1) Compared with MEF cells transfected with negative control miRNA, the expression levels of Ulk1 protein and mRNA in MEF cells transfected with pre-miR-93 were decreased whereas they were relatively more abundant in MEF cells transfected with anti-miR-93. (2) Through ?-Gal reporter gene assay, miR-93 was proved to interact with the predicted binding sites on the 3'UTR of Ulk1 mRNA. Since Ulk1 is an essential Atg gene which forms a complex with mammalian orthologue of Atg13 and FIP200 during the initiation of autophagosome formation, miR-93 places its negative regulatory effect on autophagy initiation through suppression of Ulk1 expression. In summary, this study reveals a new negative feedback loop mechanism between autophagy and miR-93. Data from this study provided new evidence that certain miRNAs could play important roles in autophagy via regulation of key Atg genes such as Ulk1. Understanding such a novel mechanism is beneficial for the development of miRNA-based therapeutics against various autophagy related diseases through modulation of autophagy.
Appears in Collections:Master's Theses (Open)

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