Construction and identification of a HEK293 cell line with stable TrxR1 overexpression.
10.12122/j.issn.1673-4254.2022.04.11
- Author:
Xiao Mei LYU
1
;
Zhi Yin ZHOU
1
;
Li ZHU
1
;
Ji ZHOU
2
;
Hui Dan HUANG
1
;
Chao ZHANG
1
;
Xiao Ping LIU
1
Author Information
1. Center of Drug Screening and Evaluation, Wannan Medical College, Wuhu 241000, China.
2. Center for Reproductive Medicine, First Affiliated Hospital of Wannan Medical College, Wuhu 241000, China.
- Publication Type:Journal Article
- Keywords:
cell model;
lentiviral packaging;
overexpression;
thioredoxin reductase 1
- MeSH:
Auranofin;
Cell Line, Tumor;
Genetic Vectors;
HEK293 Cells;
Humans;
Lentivirus/genetics*;
RNA, Messenger;
Transfection
- From:
Journal of Southern Medical University
2022;42(4):554-560
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.
METHODS:TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.
RESULTS:TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).
CONCLUSION:We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.
