Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB1 exposed model

doi:10.3969/j.issn.1004-1524.20230325

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (2): 272-283.DOI: 10.3969/j.issn.1004-1524.20230325

• Animal Science • Previous Articles Next Articles

Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model

WANG Lin¹^,²(), YUAN Jianlin¹^,², MIAO Chang¹^,², MA Yuhan¹^,², CAO Sanjie¹^,², ZHAO Qin¹^,²^,^*()

1. Research Center for Swine Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique, Ministry of Agriculture and Rural Affairs, Chengdu 611130, China

Received:2023-03-14 Online:2024-02-25 Published:2024-03-05

Abstract

Abstract:

To construct three LO2 cell lines separately with POR gene stable knockout (POR^KO), complementation (POR^CO), and overexpression (POR^OE), and provide cell models for further in-depth research on the function of the POR gene in the mechanism of AFB₁toxicity. Firstly, two sgRNAs targeting the POR gene were designed using CRISPR/Cas9 technology and cloned into the lentiCRISPR v2 vector, and then lentivirus packaging and virus infection were performed. After screening LO2 POR^KO monoclonal cells, they were sequenced and identified by Western blot. Secondly, the pcDNA3.1(+)/myc-His B-POR recombinant plasmid was constructed by homologous recombination method, and the LO2 POR^CO and LO2 POR^OE cell lines were respectively constructed through cell transfection and were identified by Western blot after screening with G-418. After the wild-type LO2, LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines were exposed with 4 μmol·L^-1 and 8 μmol·L^-1 AFB₁for 48 h, then the cell morphology was observed, and the cell viability was determined. The results showed that sgRNA-2 could successfully knock out the POR gene to obtain the LO2 POR^KO cell line, and the LO2 POR^CO and LO2 POR^OE cell lines were successfully constructed. After each cell line was exposed to 4 μmol·L^-1 and 8 μmol·L^-1 AFB₁for 48 h, compared with the wild-type LO2 cell viability rate of 43.66%±0.58% and 27.90%±0.46%, the cell viability rate of LO2 POR^KO cells was extremely significant, both 100% (P<0.000 1); the cell viability rate of LO2 POR^CO cells was still significantly different, respectively 57.07%±0.74% and 42.54%±0.68% (P<0.05); the cell viability rate of LO2 POR^OE cells was significantly lower, respectively 24.58%±0.92% and 17.99%±0.81% (P<0.01). In conclusion, this paper successfully constructed LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines, and studied the cell viability rate and cell morphology changes of normal LO2, LO2 POR^KO, LO2 POR^CO, and LO2 POR^OE cell lines with AFB₁exposure, and discovered that the POR gene has a huge impact on AFB₁-induced cytotoxicity. This paper laid a foundation for the subsequent in-depth research on the function of the POR gene in the mechanism of AFB₁toxicity.

Key words: POR, CRISPR/Cas9, LO2 cells, AFB₁

CLC Number:

S851.31

WANG Lin, YUAN Jianlin, MIAO Chang, MA Yuhan, CAO Sanjie, ZHAO Qin. Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 272-283.

Figures/Tables 11

Table 1 sgRNA and corresponding primer sequences

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

Table 2 POR gene deletion identification primer sequence

靶位点 Target	敲除鉴定引物序列 Primer sequence(5'→3')
sgRNA-POR-1	hPOR-KO1-F: AACCTTGAACAGGCTCAGTCAT hPOR-KO1-R: ATACATGGATCTGGAGTCCCC
sgRNA-POR-2	hPOR-KO2-F: AGCCCCTCCGTGTTGTTA hPOR-KO2-R: AGGGGACATTCTCGTAGTGC

Table 3 POR homologous amplified primer sequence

引物名称 Primer name	引物序列 Primer sequence(5'→3')
POR-F	ctaccggactcagatctcgagATGATCAACATGGGAGACTCCC
POR-R	gtaccgtcgactgcagaattcCTAGCTCCACACGTCCAGGG

Fig.1 Construction of lentiCRISPR v2-sgRNA-POR recombinant plasmid A, lentiCRISPR v2 plasmid information partial schematic diagram and sgRNA insertion location; B, sgRNA sequence and target site diagram; C, lentiCRISPR v2-sgRNA recombinant plasmid sequencing results.

Fig.2 Identification of LO2 PORKO monoclonal cell lines A, LO2 PORKO cell sequencing peak map; B, LO2 PORKO cell sequence comparison map.

Fig.3 Construction of pcDNA3.1(+)/myc-His B-POR recombinant eukaryotic expression plasmid A, pcDNA3.1(+)/myc-His B plasmid partial information schematica and insertion site of POR target fragment; B, POR homologous fragment amplified in LO2 cell cDNA as template,1 is DNA marker,2 is the target fragment; C, PCR electrophoretic map of colony identification, 1 is DNA marker, 2-4 is positive colony; D, Sequencing results of positive colonies.

Fig.4 Results of WB identification of cell lines With LO2 cells as the control, WB detected the expression of POR protein in LO2 PORKO, LO2 PORCO and LO2 POROE cells, and the amino acid sequences in LO2 PORKO cells were analyzed.

Fig.5 The toxic dose-effect curve of AFB1on the relative cell viability of wild-type LO2 cell lines

Table 4 The results of IC values

毒素IC值 Toxin IC value		浓度的95%置信限度 95% confidence limit for concentration/(μmol·L^-1)			毒素IC值 Toxin IC value		浓度的95%置信限度 95% confidence limit for concentration/(μmol·L^-1)
毒素IC值 Toxin IC value		估算 Estimated value	上限 Upper limit	下限 Lower limit	毒素IC值 Toxin IC value		估算 Estimated value		上限 Upper limit		下限 Lower limit
AFB₁	IC₁₀	1.57	0.00	4.44	AFB₁	IC₁₀	0.22	0.02		0.65
24 h	IC₁₅	4.97	0.10	9.37	48 h	IC₁₅	0.41	0.05		1.03
	IC₂₀	11.43	3.34	25.27		IC₂₀	0.66	0.12		1.46
	IC₂₅	23.11	12.68	270.59		IC₂₅	0.97	0.22		1.94
	IC₃₀	42.74	21.06	4.14×10³		IC₃₀	1.36	0.37		2.50
	IC₃₅	74.72	30.47	5.41×10⁴		IC₃₅	1.86	0.61		3.15
	IC₄₀	126.03	42.02	6.14×10⁵		IC₄₀	2.48	0.96		3.92
	IC₄₅	208.04	56.62	6.37×10⁶		IC₄₅	3.27	1.49		4.86
	IC₅₀	339.97	75.45	6.33×10⁷		IC₅₀	4.28	2.26		6.05
	IC₆₀	917.09	133.59	6.62×10⁹		IC₆₀	7.41	5.02		9.89
	IC₇₀	2 704.23	247.31	1.06×10¹²		IC₇₀	13.45	10.07		20.06
	IC₈₀	1.01×10⁴	521.74	5.18×10¹⁴		IC₈₀	27.86	18.96		59.05
	IC₉₀	7.36×10⁴	1.60×10³	5.80×10¹⁸		IC₉₀	83.30	43.69		336.79
	IC₉₉	2.60×10⁴	4.30×10⁴	5.52×10³⁰		IC₉₉	2.12×10³	465.01		6.44×10⁴

Fig.6 Effect of morphological characteristics on cells after 48 h treated with AFB1under optical microsope (100×)

Fig.7 Effect of AFB1treatment on cell viability The cell viability of LO2, LO2 PORKO, LO2 PORCO and POROE cells treated with AFB1for 48 hours. Different cell lines at different concentrations were compared with negative control groups, **, P<0.01; ***, P<0.001; ns, P>0.05. Compared with LO2 cell group under the same concentration of AFB1, *, P<0.05; **, P<0.01; ****, P<0.000 1.

References 31

[1]	LI Y, MOU Y, THUNDERS M, et al. Effects of enrofloxacin on antioxidant system, microsomal enzymatic activity, and proteomics in porcine liver[J]. Journal of Veterinary Pharmacology and Therapeutics, 2018, 41(4): 562-571.
[2]	GONG L, ZHANG C M, LV J F, et al. Polymorphisms in cytochrome P450 oxidoreductase and its effect on drug metabolism and efficacy[J]. Pharmacogenetics and Genomics, 2017, 27(9): 337-346.
[3]	UNAL E, DEMIRAL M, YıLDıRıM R, et al. Cytochrome P450 oxidoreductase deficiency caused by a novel mutation in the POR gene in two siblings: case report and literature review[J]. Hormones, 2021, 20(2): 293-298.
[4]	XIANG P, HE R W, LIU R Y, et al. Cellular responses of normal (HL-7702) and cancerous (HepG2) hepatic cells to dust extract exposure[J]. Chemosphere, 2018, 193: 1189-1197.
[5]	RECZEK C R, BIRSOY K, KONG H, et al. A CRISPR screen identifies a pathway required for paraquat-induced cell death[J]. Nature Chemical Biology, 2017, 13(12): 1274-1279.
[6]	PEDERSEN M H, HOOD B L, EHMSEN S, et al. CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients[J]. International Journal of Cancer, 2019, 144(3): 631-640.
[7]	MARCHESE S, POLO A, ARIANO A, et al. Aflatoxin B1 and M1: biological properties and their involvement in cancer development[J]. Toxins, 2018, 10(6): 214.
[8]	彭双清, 郝卫东, 伍一军. 毒理学替代法[M]. 北京: 军事医学科学出版社, 2009.
[9]	GUPTA D, BHATTACHARJEE O, MANDAL D, et al. CRISPR-Cas9 system: a new-fangled dawn in gene editing[J]. Life Sciences, 2019, 232: 116636.
[10]	MILLER W L, WHITE P C. History of adrenal research: from ancient anatomy to contemporary molecular biology[J]. Endocrine Reviews, 2023, 44(1): 70-116.
[11]	REED L, JARVIS I W H, PHILLIPS D H, et al. Enhanced DNA adduct formation by benzo[a]pyrene in human liver cells lacking cytochrome P450 oxidoreductase[J]. Mutation Research Genetic Toxicology and Environmental Mutagenesis, 2020, 852: 503162.
[12]	HEINTZE T, KLEIN K, HOFMANN U, et al. Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells[J]. Scientific Reports, 2021, 11(1): 1000.
[13]	REED L, JARVIS I W H, PHILLIPS D H, et al. Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells[J]. Mutagenesis, 2019, 34(5/6): 413-420.
[14]	LI L J, YE T, ZHANG Q Y, et al. The expression and clinical significance of TPM4 in hepatocellular carcinoma[J]. International Journal of Medical Sciences, 2021, 18(1): 169-175.
[15]	HAN Y Y, XIAO K J, TIAN Z X. Comparative glycomics study of cell-surface N-glycomes of HepG2 versus LO2 cell lines[J]. Journal of Proteome Research, 2019, 18(1): 372-379.
[16]	SONG Z Y, WU F L, ZHENG Y T, et al. Cellular toxicity study of silicon nanowires[J]. Dose-Response: a Publication of International Hormesis Society, 2020, 18(2): 1559325820918761.
[17]	BAI C Z, HAO J Q, HAO X L, et al. Preparation of Astragalus membranaceus lectin and evaluation of its biological function[J]. Biomedical Reports, 2018, 9(4): 345-349.
[18]	JANIK E, NIEMCEWICZ M, CEREMUGA M, et al. Various aspects of a gene editing system-CRISPR-Cas9[J]. International Journal of Molecular Sciences, 2020, 21(24): 9604.
[19]	ZHANG C, QUAN R F, WANG J F. Development and application of CRISPR/Cas9 technologies in genomic editing[J]. Human Molecular Genetics, 2018, 27(R2): R79-R88.
[20]	WANG H F, NAKAMURA M, ABBOTT T R, et al. CRISPR-mediated live imaging of genome editing and transcription[J]. Science, 2019, 365(6459): 1301-1305.
[21]	MEMI F N, NTOKOU A, PAPANGELI I. CRISPR/Cas9 gene-editing: research technologies, clinical applications and ethical considerations[J]. Seminars in Perinatology, 2018, 42(8): 487-500.
[22]	耿梦雅, 王李卓, 章尧, 等. 溶酶体膜蛋白Sidt2缺失导致肝脏细胞自噬受损[J]. 南方医科大学学报, 2021, 41(8): 1207-1213.
	GENG M Y, WANG L Z, ZHANG Y, et al. Lysosomal membrane protein Sidt2 deletion impairs autophagy in human hepatocytes[J]. Journal of Southern Medical University, 2021, 41(8): 1207-1213. (in Chinese with English abstract)
[23]	HENDRIKS D, ARTEGIANI B, HU H L, et al. Establishment of human fetal hepatocyte organoids and CRISPR-Cas9-based gene knockin and knockout in organoid cultures from human liver[J]. Nature Protocols, 2021, 16(1): 182-217.
[24]	LV H M, HONG L H, TIAN Y, et al. Corilagin alleviates acetaminophen-induced hepatotoxicity via enhancing the AMPK/GSK3β-Nrf2 signaling pathway[J]. Cell Communication and Signaling, 2019, 17(1): 2.
[25]	LEE H K, LIM H M, PARK S H, et al. Knockout of hepatocyte growth factor by CRISPR/Cas9 system induces apoptosis in hepatocellular carcinoma cells[J]. Journal of Personalized Medicine, 2021, 11(10): 983.
[26]	霍桂桃, 文海若, 吕建军, 等. 药物毒理学研究中体外替代试验研究现状及展望[J]. 药物评价研究, 2018, 41(12): 2133-2141.
	HUO G T, WEN H R, LÜ J J, et al. Current situation and prospects of in vitro alternative tests in drug toxicological study[J]. Drug Evaluation Research, 2018, 41(12): 2133-2141. (in Chinese with English abstract)
[27]	RUSHING B R, SELIM M I. Aflatoxin B1: a review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods[J]. Food and Chemical Toxicology: an International Journal Published for the British Industrial Biological Research Association, 2019, 124: 81-100.
[28]	FAN T T, XIE Y L, MA W B. Research progress on the protection and detoxification of phytochemicals against aflatoxin B₁-Induced liver toxicity[J]. Toxicon: Official Journal of the International Society on Toxinology, 2021, 195: 58-68.
[29]	LIU W Y, WANG L Q, ZHENG C F, et al. Microcystin-LR increases genotoxicity induced by aflatoxin B1 through oxidative stress and DNA base excision repair genes in human hepatic cell lines[J]. Environmental Pollution, 2018, 233: 455-463.
[30]	ZHU Q, MA Y R, LIANG J B, et al. Correction: AHR mediates the aflatoxin B1 toxicity associated with hepatocellular carcinoma[J]. Signal Transduction and Targeted Therapy, 2021, 6(1): 432.
[31]	PAULETTO M, TOLOSI R, GIANTIN M, et al. Insights into aflatoxin B1 toxicity in cattle: an in vitro whole-transcriptomic approach[J]. Toxins, 2020, 12(7): 429.

Construction of POR gene knockout, complementation and overexpression LO2 cell lines and preliminary application as AFB₁ exposed model

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