Date of Award

2020

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmacology

First Advisor

Janet L. Stein

Abstract

The overall 5-year survival rate for woman diagnosed with breast cancer has increased significantly over the last 20 years. However, prognosis for women with stage IV, metastatic disease remains very poor. Women diagnosed with stage 0-III breast cancer have above an 85% chance of survival over a 5-year period while women diagnosed with stage IV breast cancer have a 5-year survival of less than 30%. A better understanding of the molecular mechanisms driving aggressive breast cancer is essential for the potential discovery of more targeted therapies to increase the survival rates for women diagnosed with stage IV breast cancer.

Previous members of the Stein/Lian laboratory profiled 113 novel, small RNA molecules in various breast cancer cell models to identify potential candidates for testing. In this study, we examine a novel class of short, non-coding RNA molecules called tRNA-derived small RNAs (tsRNAs). These molecules are 18-48 nucleotides long and are cleaved RNA fragments generated during tRNA maturation. We identified two candidate tsRNAs, ts-2 and ts-112, that have relatively low expression in the normal-like breast epithelial cell line, MCF10A, and increase in expression as breast cancer becomes more aggressive, with the highest expression in the MCF10CA1a cell line.

Because ts-2 and ts-112 are upregulated in MCF10CA1a cells compared to MCF10A cells, we hypothesized that ts-2 and ts-112 promote breast cancer disease progression. In this study, inhibitors of ts-2 and ts-112 were transfected into MCF10CA1a cells and mimics were transfected into MCF10A cells to further understand the role of these molecules in certain cancer phenotypes. Assays were performed to assess the affect of tsRNA on cell growth, death, migration, invasion, and any cell cycle changes after ts-2 and ts-112 gain or loss of function

We observed a decrease in cell growth rate over time after ts-2 and ts-112 inhibition in MCF10CA1a cells. In trypan blue exclusion growth curve assays, tsRNA inhibited populations had fewer cells than mock and negative control populations over a 72-hour time course. Further, we observed parallel results with CCK-8 assays for cell proliferation and in a flow cytometry analysis of the cell cycle. The percent of cells in S phase increased in the ts-2 and ts-112 inhibited populations when compared to the mock and negative control, indicating that cells are blocked in S phase after tsRNA inhibition. We observed an increase in cell growth rate over time after ts-2 and ts-112 overexpression in MCF10A cells. We performed trypan blue exclusion growth curve assays and saw an increase in population growth over a 72-hour time course. In parallel, similar results were observed using a CCK-8 cell proliferation assay, where ts-2 and ts-112 overexpressed populations had an increase in cell proliferation compared to mock and negative control. Cell death, migration and invasion were unchanged when ts-2 and ts-112 levels were manipulated in these cell models.

Our results reveal the role of ts-2 and ts-112 in breast cancer and provide a better understanding of the molecular mechanisms related to a worsened prognosis for women with stage IV disease compared to women with stage 0-III disease. Future studies could potentially lead to the use of tsRNAs as blood biomarkers for cancer risk and diagnosis or as a target for novel chemotherapies for cancer treatment.

Language

en

Number of Pages

76 p.

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