Date on Master's Thesis/Doctoral Dissertation

12-2019

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Degree Program

Physiology and Biophysics, PhD

Committee Chair

Galandiuk, Susan

Committee Co-Chair (if applicable)

Schuschke, Dale

Committee Member

Bhatnagar, Aruni

Committee Member

Joshua, Irving

Committee Member

Kalbfleisch, Theodore

Committee Member

Maldonado, Claudio

Author's Keywords

Colon cancer; metastasis; epithelial-to-mesenchymal transition; EMT

Abstract

Colorectal adenocarcinoma is the fourth most common cancer diagnosed worldwide and is a significant cause of morbidity and mortality. This dissertation performed an exploratory RNA-sequencing analysis comparing gene expression between colon adenocarcinoma tissue and paired normal colon epithelium. After identification of a number of lncRNAs that were increased in expression in colon adenocarcinoma compared to normal colon epithelium, we aimed to validate the expression and investigate their function in vitro. Specifically, we focused on the lncRNA ZFAS1 and its association with epithelial-to-mesenchymal transition. These studies found the following: 1. Seven candidate lncRNAs were identified from the exploratory RNA-sequencing analysis to be significantly increased in expression in colon adenocarcinoma, three of which ZFAS1, GAS5, and PVT1 were found to be significantly increased in colon adenocarcinoma compared to paired normal colon epithelium as examined by laser capture microdissection. 2. Both ZFAS1 and GAS5 are significantly increased in cytoplasm of cell lines compared to the nucleus, whereas PVT1 was more represented in the nucleus. As such there was significant knockdown of both ZFAS1 and GAS5 following transfection with siRNA. 3. Knockdown of ZFAS1 leads to decreased proliferation and migration in colon adenocarcinoma cell lines. In contrast, knockdown of GAS5 did not lead to a change in proliferation. We focused our subsequent investigation on ZFAS1. 4. ZFAS1 has a reciprocal relationship with miR-200b and miR-200c expression in vitro but not with three of the other experimentally verified miRNAs that bind ZFAS1. We also validated the functional effect of miR-200b and miR-200c mimics on decreasing cell migration. 5. ZFAS1 knockdown is associated with the functional changes on cellular phenotype through decreasing ZEB1 expression through miR-200 signaling, causing a subsequent increase in the expression of the epithelial marker, E-cadherin, and a decrease in the expression of the mesenchymal marker, vimentin. These findings demonstrate an association between ZFAS1 and miR-200/ZEB1/E-cadherin, vimentin signaling in EMT signaling in colon adenocarcinoma. In contrast to typical EMT signaling, ZFAS1 knockdown also leads to decreased cell proliferation suggesting its potential value as a therapeutic agent.

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