INVESTIGATING THE ROLE OF PRDX1 IN REGULATING THE METALLOTHIONEIN GENES IN DETOXIFYING HEAVY METALS

Abstract

Background: PRDX1 is encoded by the peroxiredoxin 1 gene, which balances the free radicals in the cells by decomposing H2O2. It is well known that PRDX1 has a role in protecting the cells from oxidative stress and preventing carcinogenesis. Previous preliminary data from our laboratory revealed that shRNA downregulation of the PRDX1 gene led to a decreased expression of several metallothionein genes as detected by microarray analysis. In this study, we aimed to investigate the role of PRDX1 in regulating the metallothionein genes by using complete PRDX1 knockout HeLa cells. Material and Methods: We used qRT-PCR and western blot to verify the cells lacking expression of the PRDX1 gene. In addition, qRT-PCR was conducted to assess the expression of the metallothionein genes in the HeLa wild-type and PRDX1 knockout cells. Since metallothioneins are required to chelate metal ions, we first set up a standard clonogenic assay to determine (IC50) of arsenite for the wild-type cells to establish conditions to test various cell types deleted for the PRDX1 gene. Results: Our results showed that PRDX1 knockout HeLa cells did not express PRDX1 at the mRNA and protein levels. In addition, we demonstrated that in the absence of PRDX1 in HeLa cells, the metallothionein genes, including MT1X, MT1G, and MT1H were significantly downregulated when compared to the HeLa wild-type cells. In parallel with HeLa cells, HEK293 cell lines were successfully transfected with CRISPR-Cas9 gRNA plasmids to delete the PRDX1 gene. Our editing assay with T7EI revealed the highest editing efficiency in HEK293 cells transfected with gRNA2 compared to gRNA1 or gRNA3. Conclusion: Our study demonstrated that although PRDX1 knockout HeLa cells were not sequenced to identify the mutation, they did not express the PRDX1 gene and PRDX1 protein. Moreover, we confirmed the findings of previous microarray data that used shRNA knockdown HeLa cells by reporting the downregulation of metallothionein genes in PRDX1 null cells. Further experiments are needed to check if the PRDX1 knockout HEK293 cells yield a similar phenotype as the HeLa cells and whether they will be sensitive to arsenite.

Date of Award	2022
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences