Date of Award

1-1-2012

Document Type

Campus Access Dissertation

Department

Biological Sciences

First Advisor

Dan A Dixon

Abstract

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in the United States and a leading cause of cancer morbidity. The initiation and progression of colorectal tumorigenesis is characterized by the accumulation of genetic alterations that allow for uncontrolled gene expression and cellular responses that lead to the development of cancer. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as essential mediators of post-transcriptional gene regulation and are being increasingly recognized for their role in cancer. MiRNA expression is dysregulated in colon cancer and these changes in miRNA expression patterns account for mis-regulation of its target mRNAs, many of which control cancer-associated pathways. Secondly, disease associated single nucleotide polymorphisms (SNPs) located in the miRNA sequence or binding site can disrupt miRNA-target interaction leading to dysregulated expression of the targeted transcript.

MiRNA-mediated regulation of genes involved in the prostaglandin pathway are of particular interest given their dysregulated expression observed in colon cancer. Alterations in expression patterns of these genes lead to dysregulated synthesis of the major end-product of this pathway, prostaglandin E2 (PGE2), which can initiate numerous signaling pathways shown to play roles in neoplastic transformation of the colon. Cyclooxygenase-2 (COX-2) is an immediate early response gene that is overexpressed in >90% of colorectal adenocarcinomas and can potentiate tumorigenesis through increased synthesis of PGE2. Conversely, inactivation of the tumor suppressor hydroxyprostaglandin dehydrogenase 15-(NAD) (15-PGDH) in colon tumors, which acts to catabolize various prostaglandins, can lead to decreased levels of PGE2, indicating a coordinated regulatory mechanism allowing for elevated PGE2 levels during colon carcinogenesis. Understanding the mechanisms by which these genes are dysregulated in cancer is of great interest, however miRNA-mediated regulation of these genes is largely unstudied.

In chapter one, I demonstrate that miR-542-3p can regulate COX-2 mRNA and protein levels through promoting rapid COX-2 mRNA decay. MiR-542-3p-mediated attenuation of COX-2 leads to decreased PGE2 levels, almost to the level of a selective COX-2 inhibitor. The T8473C SNP is located within the COX-2 3'UTR and within miR-542-3p's target binding site and is associated with increased risk for multiple cancer etiologies, including colon cancer. The T8473C SNP is sufficient to disrupt miR-542-3p-mediated regulation of COX-2 allowing for pathogenic COX-2 overexpression in colon cancer patients who harbor this polymorphism.

In chapter two, I describe how the tumor suppressor miR-16 can also regulate COX-2 expression, and that inhibition of miR-16 within the intestinal epithelium can alter COX-2 and other miR-16 target gene expression, including multiple genes involved in cell cycle maintenance. The use of anti-miR oligonucleotides (AMOs) has been studied using various chemical modifications and delivery modalities, and we show that oral gavage delivery of 2'-O-methoxyethyl (2'MOE) modified AMO-miR-16 is capable of attenuating miR-16 levels in the murine gastrointestinal tract. Consistent treatment with AMO-miR-16 in ApcMin/+ mice is efficacious in promoting increased tumorigenesis within the small intestine and colon, and is associated with dysregulation of miR-16 target genes and associated pathological response.

Elevated levels of PGE2 observed in colon cancer have been well demonstrated to occur through a COX-2-dependent mechanism. However, simultaneous loss of the PGE2-catabolizing enzyme, 15-PGDH, in colonic tumors has also been shown to contribute to increased prostaglandin levels and poor patient survival. In chapter three, I illustrate that the most upregulated miRNA in colon tumors, miR-21, and 15-PGDH are inversely correlated in colon cancer patients and that miR-21 can directly regulate 15-PGDH expression, allowing for decreased PGE2 levels in colon cancer cells. In vivo inhibition of miR-21 by use orally delivered modified AMOs against the mature miR-21 sequence allows for elevated 15-PGDH expression and correlating PGE2 levels in the gastrointestinal tract, indicating an alternative mechanism of miRNA-mediated regulation of PGE2 levels.

Finally, I demonstrate in chapter four the ability of miR-542-3p to control other physiological responses independent of COX-2 inhibtion and examine the role of miR-542-3p in regard to its role in mediating cellular apoptosis. miR-542-3p induces apoptosis in a p53-dependent manner by promoting increased p53 protein stability and accumulation allowing for activation of multiple down-stream p53 target genes involved in the apoptotic response. Taken together, these findings demonstrate that disruption of miRNA function in colon cancer can occur either by changes in miRNA expression patterns or by impacting miRNA targeting ability and leads to dysregulation of target gene expression involved in colon tumorigenesis.

Rights

© 2012, Ashleigh Eleanor Moore

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