Date on Master's Thesis/Doctoral Dissertation

12-2016

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)

Joshua, Irving

Committee Member

Bhatnagar, Aruni

Committee Member

Maldonado, Claudio

Committee Member

Rai, Shesh

Committee Member

Schuschke, Dale

Abstract

This dissertation investigated the role of the miR-200 family in normal colon epithelial (CCD 841) and Dukes’ C (HT-29) colorectal cancer (CRC) cell lines. Our aim was to characterize expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) in colorectal cell lines, study their effect on the tumor suppressor Ras Associated Domain-Containing Protein (RASSF) 2 and on subsequent activity within the mitogen-activated protein kinase (MAPK) signaling pathway. We wanted to determine whether regulation of miR-200 family members could change cell behavior towards more “cancer-like” in a normal colon epithelium (CCD 841) cell line, or less “cancer-like” in a Dukes’ C (HT-29) CRC cell line. We found the following: 1. All miR-200 family members were highly expressed in colorectal cancer cell lines compared to a normal colon epithelial cell line. 2. RASSF2 mRNA and protein expression was downregulated in all CRC cell lines compared to the normal colon epithelial (CCD 841) cell line. 3. Overexpression of miR-200 family members in a normal colon epithelial (CCD 841) cell line decreased expression of both RASSF2 mRNA and protein. 4. Inhibition of miR-200 family members in a Dukes’ C (HT-29) CRC cell line increased expression of both RASSF2 mRNA and protein. 5. Total K-Ras expression and phosphorylation of ERK 1/2 increased following overexpression of miR-200 family members in a normal colon epithelial (CCD 841) cell line, indicating increased activity within the MAPK pathway resulting in increased cell proliferation. 6. MAPK pathway activity decreased, as measured by reduced ERK 1/2 phosphorylation and reduced cell proliferation in a Dukes’ C (HT-29) CRC cell line following inhibition of miR-200 family members. These findings demonstrate a novel association of the miR-200 family, the tumor suppressor RASSF2, and the MAPK signaling pathway in CRC. In contrast to the previous understanding that miR-200 family dysregulation is considered to exhibit tumor suppressive behavior by blocking epithelial to mesenchymal transition, we refute this in the case of CRC and propose the miR-200 family contribute to CRC tumorigenesis. This improved understanding of the miR-200 family may have the potential to be developed as a therapeutic intervention in CRC.

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