Date of Award

Spring 5-5-2015

Degree Type

Honors Thesis

Major

Biology

Degree Name

Bachelor of Science

Department

Biology

First Advisor

Dr Juliet V. Spencer

Second Advisor

Dr Gary L. Stevens

Third Advisor

Dr James Sikes

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous virus that infects 70-90% of the general population, primarily the immunocompromised, but has been implicated in several forms of cancer, including breast cancer. Breast cancer is the second leading cause of cancer related deaths in women in North America, usually from metastasis. Exosomes are 30-100nm vesicles produced by most cells which carry protein and RNA to cells in their microenvironment. The aim of this study is to investigate the impact of HCMV-infection of a secreted viral cytokine, cmvIL-10, on exosome production by highly metastatic breast cancer cells.

MDA-MB-231 cells were cultured in vitro, and were treated with cmvIL-10. Exosomes were isolated from cell media via ultracentrifugation. A subsequent quantification colorimetric assay, quantitative polymerase chain reaction (qPCR), Western Blot and fluorescent tagging and reintroduction to untreated cells were used to determine number, content and localization of collected exosomes.

The results showed that there was a definite difference in the number of exosomes produced between MDA cells treated or not treated with cmvIL-10. There were significant quantities of exosomes produced by MDA cells treated for 72hrs, but not at earlier time points, nor in HEK293 cells at any time point, that could be measured. There was a statistically significant fold change in the amount of total RNA isolated from exosomes of MDA cells treated with cmvIL-10 (p=0.011). These data were further confirmed during the introduction of fluorescently tagged exosomes to untreated MDA cells, which demonstrated a perceptibly greater fluorescence in cells that took up exosomes treated with cmvIL-10. More exosomes per sample will need to be isolated to investigate miRNA or specific protein difference between groups; optimization of the protocol is required prior to further miRNA profiling and Western blot.

This study demonstrated that there is a difference in the quantity and content of exosomes produced by MDA-MB-231 cells following treatment with cmvIL-10. Further research may show whether these exosomic profiles can be viable biomarkers to indicate cancer and HCMV status.

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