Leveraging miRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561-C as a strong miRQTL for circulating miRNA-1908-5p (P=4.8x10-31) which has an inverse relationship with circulating LDL-C, fasting glucose and A1c. Here I investigated the molecular mechanism(s) linking miR1908-5p to cholesterol metabolism. First, by overexpression experiments in HuH-7 cells demonstrate that the presence of the C allele, associated with lower LDL-C levels, significantly increases miR-1908-5p by 2.15-fold relative to the T allele. Further experiments revealed that 72-hour cholesterol depletion increases miR-1908-5p expression (2.11-fold) whereas cholesterol loading decreases miR-1908-5p expression (0.69-fold). Differential miR-1908-5p expression was then used to profile genes involved in lipoprotein signaling and cholesterol metabolism using a PCR array to identify LDLR as a gene of interest. Although total RNA and protein expression of LDLR was unchanged in response to differential miR-1908-5p expression, the ratio of the mature form to the cleaved form of LDLR decreased following miR-1908-5p inhibition (0.85-fold) and conversely, increased with mimic treatment (1.63-fold). Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL. These findings uncover a potential mechanism linking miR-1908-5p to lower LDL-cholesterol levels through reduced LDLR cleavage.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/40422 |
| Date | 24 April 2020 |
| Creators | Beehler, Kaitlyn |
| Contributors | McPherson, Phyllis |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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