Circadian clock controls an organism’s biological rhythm and regulates physiological conditions in response to external time cues. Most of living organisms have their own time-keeping mechanism that is maintained by transcriptional-translational autoregulatory feedback loops involving several clock genes such as Periods, Cryptochromes, Clock and Bmal1. Recent discoveries have found the relevance between changes in circadian oscillation and post-transcriptional modification by microRNAs (miRNAs). However, the specific mechanisms of miRNAs on circadian oscillation still remain unclear. This study demonstrated the regulatory effects of miR-24-3p and miR-25-3p on Period2 (Per2) expression by direct interaction with 3’ untranslated region (UTR) of Per2 using luciferase reporter assay. Furthermore, real-time bioluminescence analyses demonstrated that PER2 oscillation patterns were sensitive to intracellular concentration of miR-24-3p or miR-25-3p. Elevation of either miR-24-3p or miR-25p-3p resulted phase advancing and dampening, while down regulation of miR-24-3p and miR-25-3p caused phase delay and increase in relative amplitude of PER2 oscillation. In summary, miR-24-3p and miR-25-3p are involved in fine-tuning of circadian rhythmicity through regulating PER2 oscillation in conjunction with other post-translational modulators. ⓒ 2017 DGIST
Table Of Contents
1. Introduction 7-- 2. Materials and Methods 11-- 3. Results 14-- 3.1 Functional Validation of Candidate microRNAs Targeting 3’UTR of mouse Period2 mRNA 14-- 3.2 Overexpression of miR-24-3p or miR-25-3p Downregulates PER2 Expression in Real-time Bioluminescence Analysis 15-- 3.3 Inhibition of Endogenous miR-24-3p or miR-25-3p Upregulates PER2 Expression in Real-time Bioluminescence Analysis 16-- 3.4 3’ Untranslated Region of Per2 mRNA is Essential for miR-24-3p and miR-25-3p for Modulating Circadian Oscillation 16-- 4. Discussion 38-- 5. References 42