Date of Award

Spring 5-24-2022

Degree Type

Thesis

Degree Name

Master of Science in Integrative Biology (MSIB)

Department

Biology

Committee Chair/First Advisor

Dr. Thomas McElroy

Major Professor

Dr. Eric A. Albrecht

Second Committee Member

Dr. Susan M. E. Smith

Third Committee Member

Dr. Austin Brown

Abstract

Wound and regeneration involves a series of complex processes that can induce oxidative stress by elevating intracellular reactive oxygen species (ROS) levels. In this study, we used RNA-seq and qPCR to examine the molecular signature of Human Embryonic Kidney cells (HEK-293T) stimulated with Crotalus atrox (CA) venom. NGS data was assembled and annotated utilizing the Illumina paired-end RNA-seq approach with software packages HISAT2 and StringTie. Results showed 243 genes were differentially expressed (106 upregulated, 137 downregulated) between CA venom stimulated and non-stimulated cells (p<0.05). HEK-293T stimulated with CA venom for 4 hours showed an increase in the expression of oxidative stress response genes (e.g., TOMM6, EGR (1,2,3), HSP70 (1A, 1B), SPRY4, ETV5, and EMP1) (Fold-Change (fc) >2.0). Significantly downregulated genes were associated with many novel transcripts/proteins such as AC117378, AD000671, and AC023509. Quantitative PCR (qPCR) was used to evaluate several genes identified from RNA-seq data. Cells stimulated with CA venom for 4 hours induced an up-regulation of MT1X (fc: 2.01±0.28), HSPA1B (fc: 2.20±0.35), and cytochrome P450 1A1 (CYP1A1) (fc: 3.24±1.06), matching NGS data trends. Extending CA venom stimulation to 8 hours significantly increased the gene expression of metallothionein 1X (MT1X, fc: 5.64±1.34) and peroredoxin-4 (PRDX4, fc: 3.29±0.43) compared to non-stimulated cells (p<0.05). Interestingly, pre-incubating cells with polyethylene glycol (PEG) catalase followed by stimulating cells with CA venom for 8 hours produced gene expression values that were downregulated in comparison to CA venom stimulation alone for 8 hours in all gene targets except for glutathione peroxidase-1 (GPx-1). However, pre-incubating cells with polyethylene glycol (PEG) catalase then stimulating cells with CA venom for 8 hours produced an MT1X and CYP1A1 gene expression values that were not significantly different from the non-stimulated controls. In contrast, GPx-1 was significantly downregulated (fc: 0.32±0.06) after an 8-hour stimulation with CA venom, compared to non-stimulated cells. Our data suggests CA venom induced cellular injury involves several gene families connected to oxidative stress responses.

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