CRISPR/Cas9-Based POR Knockout THLE-2 Cell Model and Its Application in AFB1 Hepatotoxicity Research

doi:10.7668/hbnxb.20196356

Abstract

Abstract:

In previous research,cytochrome P450 oxidoreductase(POR)was identified as a critical host factor facilitating aflatoxin B1(AFB1)-induced hepatotoxicity through CRISPR/Cas9 library screening.To further elucidate the mechanistic role of POR in AFB1-mediated hepatocellular damage,we designed two single-guide RNAs(sgRNAs)targeting exon 5 of the human POR gene,based on its published sequence,using CRISPR/Cas9 gene-editing technology.These sgRNAs were cloned into the lentiCRISPRv2 vector,which was subsequently packaged into lentivirus and utilized to infect THLE-2 human hepatocytes.Monoclonal cell lines were isolated by limiting dilution,and knockout efficiency was assessed at both the mRNA and protein levels using off-target sequencing,reverse transcription quantitative PCR(RT-qPCR),and Western Blot analysis.The findings indicated that V2-sgRNA-POR-1 successfully knocked down POR expression,resulting in a significant reduction at both the transcriptional and translational levels in the selected monoclonal cells.Further experiments involved treating THLE-2 wild-type cells with varying concentrations of AFB1(100,200,400,800 μmol/L)for durations of 24,48,and 72 hours.CCK-8 cell viability assays demonstrated that AFB1 toxicity to hepatocytes was significantly dependent on both concentration and exposure time,with an optimal exposure condition identified at 200 μmol/L for 48 hours[IC₅₀(48 h)=(200.55±44.49)μmol/L].Under these conditions,POR knockout cell lines exhibited marked resistance to AFB1-induced cytotoxicity,with significantly higher cell viability than wild-type cells.These findings suggest that POR plays a critical role in facilitating AFB1-mediated hepatocyte toxicity,and that POR knockout substantially enhances cellular resistance to AFB1.

Key words: Aflatoxin B1(AFB1), Cytochrome P450 oxidoreductase(POR), CRISPR/Cas9, THLE-2 cell

CLC Number:

S852.6

YU Yang, MA Yuhan, LI Jinfeng, CHEN Jiayong, WANG Dan, CHEN Xizhu, YU Miao, YUAN Jian, LOU Jianan, CAO Sanjie, ZHAO Qin. CRISPR/Cas9-Based POR Knockout THLE-2 Cell Model and Its Application in AFB1 Hepatotoxicity Research[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 222-231. doi: 10.7668/hbnxb.20196356.

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Figures/Tables 8

Tab.1 Sequences for sgRNA-POR

靶位点 Target site	靶位点序列(5'—3') Target site sequence(5'—3')	引物序列(5'—3') Primer sequence (5'—3')
sgRNA-POR-1	AGAGATCGACAACGCCCTGG	F: ACCGAGAGATCGACAACGCCCTGG
		R: AAACCCAGGGCGTTGTCGATCTCTC
sgRNA-POR-2	GCCAGAGATCGACAACGCCC	F: GACCGCCAGAGATCGACAACGCCC
		R: AAACGGGCGTTGTCGATCTCTGGC

Tab.2 POR gene deletion identification primer sequence

靶位点 Target site	引物序列(5'—3') Primer sequence (5'—3')
sgRNA-POR-1	F:AACCTTGAACAGGCTCAGTCAT
	R:ATACATGGATCTGGAGTCCCC
sgRNA-POR-2	F:AGCCCCTCCGTGTTGTTA
	R: AGGGGACATTCTCGTAGTGC

Tab.3 Primer sequences for RT-qRCR

基因名称 Gene	序列号 Accession No.	引物序列(5'—3') Primer sequence (5'—3')	扩增子/bp Amplicon	退火温度/℃ Tm
POR	NM_000941.3	F: TCTCCTAACCTACTGGTTCCTCT	182	60.5
		R: TCCTCCCCGTTTTCTTCATCT
GAPDH	NM_002046.7	F: GGAGCGAGATCCCTCCAAAAT	197	60.0
		R: GGCTGTTGTCATACTTCTCATGG

Fig.1 Construction of the lentiCRISPR-v2-sgRNA recombinant plasmid A.sgRNA-POR target site diagram;B.The model of LentiCRISPR-V2-sgRNA-POR recombinant plasmid;C.The sequence alignment results of the target sites of the recombinant plasmid.

Fig.2 Identification of POR-KO monoclonal cell lines A.Screening of purine toxin concentrations;B.Sanger sequencing results of POR gene knockout THLE-2 cell lines;C.RT-qPCR results;D.Western Blotting analysis;E.Grayscale quantification of Western Blotting results.Different lowercase letters indicate statistically significant differences at P<0.05.

Fig.3 Assessment of cell viability in THLE-2-WT cells following exposure to AFB1

Fig.4 Morphological observation of cells after 48 h exposure to AFB1(100×)

Fig.5 Cell survival rate after 48 h exposure to AFB1 Within the same treatment concentration,groups sharing the same lowercase letter are not significantly different at P<0.05.

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