Pharm.D. (six years)
Biomedical and Pharmaceutical Sciences
hepatocellular carcinoma; farnesoid X receptor; ubiquitin specific peptidase 2
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Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide and has limited treatment options. The farnesoid X receptor (FXR) is a nuclear receptor implicated in the development of HCC. FXR is highly expressed in the liver and has many essential roles including a protective function to suppress hepatocarcinogenesis. The gene ubiquitin specific peptidase 2 (USP2) has been shown to regulate target genes involved in cell proliferation, apoptosis, and tumorigenesis. The purpose of this study focused on characterizing regulation of USP2 promoter by FXR and investigated the expression of USP2 in human cancer cell line. Human liver HCC cells (HepG2 cells) were used for investigation. To characterize the regulation of the USP2 promoter, HepG2 cells were transfected and then treated with FXR agonists and antagonists. Transfections using GenJet version II were done with USP2 alone, USP2 plus FXR alpha 1 (FXRα1), USP2 plus FXR alpha 2 (FXRα2), and USP2 plus FXRα1 plus FXRα2. HepG2 cells were then treated with FXR agonists and antagonists to see the effect on USP2 promoter. Dual luciferase assays were used to measure reporter gene expression. USP2 expression has been shown to vary throughout the day according to some literature. To look at the circadian endogenous USP2 expression, HepG2 cells were plated for a minimum of 12 hours and then collected at 6 time points (2 pm, 6 pm, 10 pm, 2 am, 6 am, 10 am) over a 24-hour period. Quantitative polymerase chain reaction (qPCR) was then used to quantify messenger RNA. The results suggest that an association between FXR and the USP2 promoter exist. Treatment with FXR agonists (GW4064 and CDCA) showed increased expression of USP2 while treatment with FXR antagonist (DY 268) showed decreased expression of USP2. Dose-response studies determined the optimal concentrations of each treatment to yield the highest expression of USP2. Additionally, the results indicate that USP2 follows a circadian rhythm where expression of the gene increases and decreases at different times of the day. The data from this study allows for a better understanding of FXR signaling activation on the transcriptional expression of USP2 and the potential role in HCC pathogenesis.