Date of Award

12-1-2010

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

MO, YIN-YUAN

Abstract

Introduction: Cisplatin combination chemotherapy is the cornerstone of treatment for many cancers including head and neck cancer. These complexes bind to the DNA and form adducts such that they ultimately trigger apoptosis. Although initial platinum responsiveness is usually high, the majority of cancer patients will eventually relapse with cisplatin-resistant disease. Thus, chemotherapy resistance is a major clinical obstacle in the successful treatment of head and neck cancer. This study aims to investigate the role of microRNAs (miRNAs) in cisplatin resistance and to better understand the underlying mechanism of cisplatin resistance in the head and neck cancer UMSCC-10B/15Scells. Methods: To determine whether aberrant miRNA expression is associated with cisplatin resistance in human head and neck cancer cells, we profiled miRNA expression in the cisplatin resistant UMSCC-10B/15S and the parental line UMSCC-10B cells by real-time PCR. Luciferase reporter assays and western blot were used to confirm targets for miR-155. We also suppressed miR-155 by anti-sense locked nucleic acid (LNA) oligo to further determine the role of miR-155 in cisplatin resistance. Results: We showed that several miRNAs were altered in UMSCC-10B/15S and among them, miR-155 was the most significantly upregulated miRNA. This was also confirmed by in situ hybridization. Furthermore, ectopic expression of miR-155 was able to increase the resistance of UMSCC-10B cells to cisplatin. Our initial characterization suggested that DMTF1, a tumor suppressor that directly binds to the Arf promoter to activate its expression, may be a potential target for miR-155 because western blot revealed that DMTF1 was downregulated in the resistant UMSCC-10B/15S cells. We then cloned 1.2 kb 3'-untranslated region (UTR) of DMTF1 into a luciferase reporter and demonstrated that miR-155 was able to suppress luciferase activity by over 50%. However, we were not able to detect any effect of ectopic expression of miR-155 on DMTF1 protein expression levels for some reason. Thus, we searched for more potential targets based on in silico analysis and the literature information. These potential targets include C/EBP-β, CTR1, E2F2, JARID2 and cyclin D1. Further characterization identified cyclinD1 to be upregulated in resistant UMSCC-10B/15S cells and, moreover expression is positively regulated by miR-155, which can be blocked by anti-miR-155. Given the role of cyclin D1 in cell proliferation and drug resistance, these results suggest that miR-155 confers cisplatin resistance in part through upregulation of cyclin D1. Conclusion: These findings suggest that deregulation of miRNAs is in resistant head and neck cancer cells and miR-155 may play a causal role in cisplatin resistance through targeting the cyclin D1 gene. As a result, suppression of miR-155 may provide a strategy to overcome the resistance in head and neck cancer.

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