"POST-TRANSCRIPTIONAL REGULATION OF COX-2 BY ALTERNATIVE POLYADENYLATION AND MIRNA-MEDIATED MECHANISMS
IN LUNG CELLS"
Ashley Lorin Cornett
Interdisciplinary Biomedical Sciences Program
B.S. 2008, Ramapo College of New Jersey
Thesis Advisor: Carol S. Lutz, Ph.D.
Department of Biochemistry and Molecular Biology
Tuesday, March 25, 2014
11:00 A.M., MSB E-609B
Prostaglandins are a class of molecules that mediate cellular inflammatory responses and control cell growth. The oxidative conversion of arachidonic acid to prostaglandin H2 is carried out by two isozymes of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed, while COX-2 is transiently induced by external stimuli, such as pro-inflammatory cytokines. COX-2 is also overexpressed in numerous cancers, including lung cancer. A marked quality of COX-2 mRNA is the presence of an unusually large 3` untranslated region (UTR). The COX-2 3`UTR, having a length of approximately 2.5 kb, is the site for multiple mechanisms of post-transcriptional regulation, including but not limited to multiple poly(A) sites and multiple putative microRNA binding sites. MicroRNAs (miRNAs) are known to be responsive to various stimuli, and their patterns of expression can vary in cancer, which, in turn, can regulate gene expression of their target mRNAs. We have demonstrated here that miR-146a expression is highly downregulated in lung cancer cells as compared to non-tumorigenic lung cells. Conversely, lung cancer cells have high levels of COX-2 protein and mRNA expression. Synthetic miR-146a can specifically ablate expression of COX-2 protein and COX-2 biological activity as measured by prostaglandin production. Therefore, we propose that miR-146a downregulation contributes to the upregulation and overexpression of COX-2 in lung cancer cells. Since potential miRNA-mediated regulation is a functional consequence of alternative polyadenylation site choice, understanding the molecular mechanisms that regulate COX-2 mRNA alternative polyadenylation and miRNA targeting will gives us key insights into how COX-2 expression is involved in the development of a metastatic condition. This thesis examined how post-transcriptional mechanisms work in concert to regulate COX-2 expression, and may provide further insight to how miRNAs can function as alternative treatment options for cancer therapy.
The latter part of this thesis examined acute induction of both COX-2 and miR-146a expression in response to cytokine stimulation. COX-2 expression can be transiently induced by various stimuli including tumor necrosis factor (TNF-ƒÑ) and interleukin-1 (IL-1ƒÒ). Similarly, miR-146a expression can be activated by lipopolysaccharide (LPS) and other proinflammatory cytokines (reviewed in Rusca and Monticelli, 2011). We propose a synergistic relationship between expression of COX-2 and miR-146a in response to cytokine treatment over time, which represents a potential model for regulation of COX-2 to be rapidly turned off upon resolution of the immune response.
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