Date of Award

Spring 5-1-2024

Document Type

Thesis

College

College of Arts and Sciences

Degree Program

Biology

Degree Name

Master of Science

Thesis Advisor

Dr. Daniel Stovall, Ph.D.

Committee Member

Dr. Laura Glasscock, Ph.D.

Committee Member

Dr. Kathryn Kohl, Ph.D.

Keywords

GBM, RYBP, miRNAs, cell viability, protein expression, PRC1, PRC2, microRNA-inhibitors, and chromatin remodeling

Abstract

Glioblastoma multiforme (GBM) is a highly aggressive and invasive tumor of the central nervous system (CNS). Survival rates are abysmal, with only 7.2% of patients alive 5-years after diagnosis. Because of this, understanding epigenetic alterations that give GBM tumors their aggressive phenotypes is critical for the development of more targeted and effective therapies. These alterations frequently affect a group of proteins called the Polycomb group proteins, which play important oncogenic and tumor suppressive roles in cancer. One Polycomb protein, the RING1- and YY1-binding protein (RYBP), is downregulated in a majority of GBM patients, suggesting a strong tumor suppressive property. In cancer, microRNAs (miRNAs) are known to target and inhibit the synthesis of tumor suppressive transcripts through binding at the mRNA’s 3’-UTR. We hypothesized that miRNAs may contribute to RYBP suppression in GBM cells. We analyzed the RYBP 3’-UTR for putative binding sites of miRNAs known to be oncogenic in GBM. We predicted that inhibition of these miRNAs would increase RYBP mRNA and protein levels, thereby decreasing GBM cell viability. To measure the effects of targeted miRNA inhibition we transfected T98G and U87MG GBM cells with synthetic miRNA-inhibitors and visualized changes in RYBP mRNA and protein expression via RT-qPCR and Western blot, respectively, and measured changes in cell viability with colorimetric WST-1 assays. Through targeted inhibition of specific miRNAs with synthetic miRNA-inhibitors we observed an increase in RYBP protein, but not mRNA levels. Surprisingly, inhibition of these specific microRNAs did not affect GBM cell viability. In fact, we actually observed a trending increase in GBM cell viability following inhibition of oncogenic miRNAs, although the increase was statistically insignificant. Overall, our study suggests RYBP is likely targeted by miRNAs in GBM. However, we found that it is possible RYBP does not mediate effects on GBM cell viability, but rather may influence other oncogenic networks, such as cell invasion and metastasis.

Comments

The King Fellowship, Jim and Karen Johston Endowment, and SC INBRE (DRP 5P20GM103499-21) for funding

Additional Files
Lee Copyright agreement.pdf (50 kB)
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