Receptor-interacting protein kinase 1 (RIPK1) is a major upstream mediator of inflammation and cell death. These processes are key to common inflammatory diseases such as atherosclerosis, where macrophages play an important role in their progression. Closely linked to the expression of downstream genes, long non-coding RNAs (lncRNAs) are critical to controlling cellular processes in health and disease. As post-transcriptional regulatory mechanisms for RIPK1 are largely unknown, this project seeks to study the stability of Ripk1 mRNA and RIPK1 protein, along with Ripk1 mRNA interactions with relevant lncRNAs under various conditions. Using transcription and translation inhibitors, we determined that both Ripk1 mRNA and RIPK1 protein are relatively unstable with half-lives of approximately 3 h. Their turnover in macrophages is further influenced by the timing and duration of inflammation. We also implemented a novel RNA pull-down procedure to capture Ripk1 mRNA and attached lncRNAs for next-generation sequencing. Through differential expression analysis, we discovered significant upregulation of known lncRNA AC125611 and novel lncRNA MSTRG.5894.1 in Ripk1-targeted samples subject to inflammation. MSTRG.7477.1 was upregulated during necroptosis, while MSTRG.5684.5 was upregulated during both inflammation and necroptosis. GapmeR-mediated knockdowns of AC125611 and MSTRG.5684.5 under inflammatory conditions resulted in decreased Ripk1 mRNA expression and RIPK1 protein expression, respectively. Meanwhile, MSTRG.7477.1 knockdowns were connected to decreased RIPK1 at both the mRNA and protein levels. Our research ultimately advances the current understanding of RIPK1 regulation by focusing on Ripk1 mRNA-lncRNA associations and turnover of its mRNA and protein in macrophages, paving the way for future investigations into their capacity to act as therapeutic targets.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/44362 |
| Date | 08 December 2022 |
| Creators | Zhou, Zier |
| Contributors | Rayner, Katey Jane |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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