Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Aldo-keto reductase 1 B10 (AKR1B10) is a secretory protein, overexpressed in human cancer cells in different cancers, such as hepatocellular carcinoma, lung squamous carcinoma, and lung adenocarcinoma in smokers. Our recent study revealed that AKR1B10 may play an oncogenic role in cancer development and progression, but little is known of its expression or regulation. In this study, we reveal a part of the regulatory mechanism by investigating the role of p53 transcription factor in regulating AKR1B10 expression level in colorectal cancer cell line, HCT-8, and hepatocellular carcinoma cell line, HepG2. In addition, we found a negative feedback loop of p53 through AKR1B10 and AMPK. This study shows that AKR1B10 promoter contains four p53 binding sites within -804bp region of the promoter which is 4kb in full length. Mutations of p53 binding sites or knockdown of p53 expression by shRNA significantly increases the basal promoter activity and the protein expression of AKR1B10. Western blot showed that overexpression of p53 protein suppresses AKR1B10 expression level. Furthermore, we demonstrate the direct interaction of p53 protein with its four binding sites ex vivo by chromatin immunoprecipitation assay. Also, EMSA assay further confirms the binding of this transcription factor protein with the first DNA binding site. Our Luciferase assay showed that p53 suppresses AKR1B10 promoter activity in a dose dependent manner. While AMPK is known to be a p53 stimulator, our lab found that AMPK can be induced by AKR1B10. Together these results suggest that p53 transcription factor downregulates AKR1B10 as a negative feedback of AMPK activity.
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