Date on Master's Thesis/Doctoral Dissertation

5-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Li, Yong

Committee Co-Chair (if applicable)

Cheng, Alan

Committee Member

Cheng, Alan

Committee Member

Clark, Barbara

Committee Member

Cole, Marsha

Committee Member

Darling, Douglas

Author's Keywords

malignant cell transformation; miR-200a; Cancer; miRNA

Subject

Cancer--Genetic aspects; Cell transformation

Abstract

Cancer is a multistep disease that begins with malignant cell transformation and frequently culminates in metastasis and death. MicroRNAs (miRNAs) are small regulatory 21-25-nt RNA molecules and are frequently deregulated in cancer. The majority of miRNAs are estimated to be co-expressed with neighboring miRNAs as clusters. Many miRNA clusters coordinately regulate multiple members of cellular signaling pathways or protein interaction networks. miR-200a is a member of the miR-200 family, which are known to be strong inhibitors of the epithelial to mesenchymal transition. As such, the tumor suppressive role of miR-200a in oncogenesis has been well studied; however, recent studies have found a proliferative role for this miRNA as well as a pro-metastatic role in the later steps of cancer progression. In this study, we employed a biphasic approach to determine miRNA involvement in malignant cell transformation. First, we screened 366 human miRNA minigenes to determine their effects on the four major cancer signaling pathways culminating in AP-1, NF-?B, c-Myc, or p53 transcriptional activity. The second phase of this study was an epithelial cell screening assay to determine the ability of miRNAs to transform epithelial cells. In our miRNA cluster profiling study, we found that miR-200a down-regulates p53 activity. miR-200a was demonstrated to directly target p53, reduce protein levels, and inhibit apoptosis. We also found that miR-200a enhances Ras-mediated transformation of MCF10A cells. Furthermore, miR-200a transforms MCF10A cells and induces tumorigenesis in immunocompromised mice by cooperating with a Ras mutant that activates the RalGEF effector pathway. These results demonstrate a role for miR-200a in malignant transformation and reveal a specific cellular environment in which miR-200a acts as an oncomiR rather than a tumor suppressor by cooperating with oncogene activation in the classical two hit model of cell transformation.

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