Date of Award

Spring 5-24-2022

Degree Type

Thesis

Degree Name

Master of Science in Integrative Biology (MSIB)

Department

Biology

Major Professor

Dr. Eric A. Albrecht

First Committee Member

Dr. Thomas McElroy

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.

Available for download on Thursday, June 06, 2024

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