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Cited by 4Liver physiology and pathologyDrug-Induced Hepatotoxicity and ProtectionPharmacogenetics and Drug Metabolism

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DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability

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Tanaka-yachi, Rieko, Aizawa, Kazuko, Shimizu, Kie, Akutsu, Hidenori, Nakamura, Kazuaki·2022·Scientific Reports
DOI 10.1038/s41598-022-25777-zPMC PMC9729623
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Abstract

HepG2 cells are widely used as a human hepatocytes model, but their functions, including drug metabolism, are inferior to primary hepatocytes. We previously reported that the hepatic gene expressions in HepG2 cells were upregulated by treatment with zebularine, which is an inhibitor of DNA methylation, through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). In this study, we established a new HepG2 cell subline, HepG2-DP cells, by stable double knockdown of DNMT1 and PKR and evaluated its function. Albumin production, expression of CYP1A2 genes, and accumulation of lipid droplets were increased in HepG2-DP cells compared with the original HepG2 cells. Comprehensive gene expression analysis of transcription factors revealed that the expression of important genes for hepatic function, such as HNF1β, HNF4α, ONECUT1, FOXA1, FOXA2, FOXA3, and various nuclear receptors, was upregulated in HepG2-DP cells. These results indicate that the newly established HepG2-DP cells are a highly functional hepatocyte cell line. In addition, we investigated whether HepG2-DP cells are able to mature by differentiation induction, since HepG2 cells are derived from hepatoblastoma. The gene expression of major CYPs and Phase II, III drug-metabolizing enzyme genes was significantly increased in HepG2-DP cells cultured in differentiation induction medium. These results suggest that HepG2-DP cells can be further matured by the induction of differentiation and could therefore be applied to studies of drug metabolism and pharmacokinetics.

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Figure 1 Morphology and lipid accumulation in HepG2-DP cells. (A) Phase-construct image of HepG2-WT and HepG2-DP cells. The cells were cultured for 72 h. Scale bar, 100 μm. (B) Oil-red O staining of HepG2-WT and HepD2-DP cells. The cells were cultured for 72 h and then fixed and stained with oil-red O. Scale bar, 40 μm. (C) Absorbance of oil red O. Absorbance at 492 nm was measured on oil red O–stained culture plates. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the unpaired t test (*p < 0.05).
Figure 1 Morphology and lipid accumulation in HepG2-DP cells. (A) Phase-construct image of HepG2-WT and HepG2-DP cells. The cells were cultured for 72 h. Scale bar, 100 μm. (B) Oil-red O staining of HepG2-WT and HepD2-DP cells. The cells were cultured for 72 h and then fixed and stained with oil-red O. Scale bar, 40 μm. (C) Absorbance of oil red O. Absorbance at 492 nm was measured on oil red O–stained culture plates. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the unpaired t test (*p < 0.05).CC BY · PubMed Central
Figure 2 Gene expression profile of transcription factors. (A) Gene expression array for hepatic transcription factors. HepG2-WT and HepD2-DP cells were cultured for 72 h. The expression level was corrected by the average value, and the data was visualized as a heat map. (B) Gene expression analysis of transcription factors by quantitative polymerase chain reaction. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the unpaired t test (*p < 0.05). (C) Immunofluorescence staining for HNF4α and FOXA1. Cells were cultured for 72 h. Immunofluorescence staining was performed to detect transcriptional factors (in red) and observed by confocal microscopy. The cell nuclei were stained with DAPI (in blue). Scale bar, 40 μm.
Figure 2 Gene expression profile of transcription factors. (A) Gene expression array for hepatic transcription factors. HepG2-WT and HepD2-DP cells were cultured for 72 h. The expression level was corrected by the average value, and the data was visualized as a heat map. (B) Gene expression analysis of transcription factors by quantitative polymerase chain reaction. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the unpaired t test (*p < 0.05). (C) Immunofluorescence staining for HNF4α and FOXA1. Cells were cultured for 72 h. Immunofluorescence staining was performed to detect transcriptional factors (in red) and observed by confocal microscopy. The cell nuclei were stained with DAPI (in blue). Scale bar, 40 μm.CC BY · PubMed Central
Figure 3 CYPs gene expression and albumin production in HepG2-DP cells. (A) Gene expression of CYP1A2 and CYP3A4. Cells were cultured for 72 h. Reverse transcription polymerase chain reaction (RT-PCR)-examined gene expression levels of CYP1A2 and CYP3A4. (B) Gene expression of albumin (ALB). Cells were cultured for 72 h. RT-PCR-examined gene expression levels of ALB. (C) Immunofluorescence staining for albumin. Cells were cultured for 72 h. Immunofluorescence staining was performed to detect albumin (in green) and observed by confocal microscopy. The cell nuclei were stained with DAPI (in blue). Scale bar, 40 μm. The exposure time of the fluorescence micrographs was adjusted to the ALB immunopositive signals of HepG2-DP cells, resulting in very weak ALB immunopositive signals in HepG2-WT at the same exposure time. (D) Albumin production of HepG2-WT and HepG2-DP cells. Albumin production per 24 h was quantified by enzyme-linked immunosorbent assay. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the t test (*p < 0.05).
Figure 3 CYPs gene expression and albumin production in HepG2-DP cells. (A) Gene expression of CYP1A2 and CYP3A4. Cells were cultured for 72 h. Reverse transcription polymerase chain reaction (RT-PCR)-examined gene expression levels of CYP1A2 and CYP3A4. (B) Gene expression of albumin (ALB). Cells were cultured for 72 h. RT-PCR-examined gene expression levels of ALB. (C) Immunofluorescence staining for albumin. Cells were cultured for 72 h. Immunofluorescence staining was performed to detect albumin (in green) and observed by confocal microscopy. The cell nuclei were stained with DAPI (in blue). Scale bar, 40 μm. The exposure time of the fluorescence micrographs was adjusted to the ALB immunopositive signals of HepG2-DP cells, resulting in very weak ALB immunopositive signals in HepG2-WT at the same exposure time. (D) Albumin production of HepG2-WT and HepG2-DP cells. Albumin production per 24 h was quantified by enzyme-linked immunosorbent assay. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the t test (*p < 0.05).CC BY · PubMed Central
Figure 4 Bile acid conjugation and transport in HepG2-DP cells. (A) Gene expression of UGT1A1 and MRP2. Cells were cultured for 72 h. Reverse transcription polymerase chain reaction–examined gene expression levels of UGT1A1 and MRP2. (B) Immunofluorescence staining of MRP2. Cells were cultured for 72 h and then fixed and stained for MRP2 (in green). The cell nuclei were stained with DAPI (in blue). Scale bar, 40 μm. (C) MRP2 transporter activity assay. CDF were accumulated in the bile canaliculus in HepG2-DP cells (Arrows). Scale bar, 40 μm. (D) Fluorescence intensity of CDF. The fluorescence intensity of CDF (Ex. 488 nm/Em. 517 nm) accumulated in the bile canaliculi was measured. The values are the mean ± standard deviation for five samples. Statistical analysis was performed by applying the t test (*p < 0.05).
Figure 4 Bile acid conjugation and transport in HepG2-DP cells. (A) Gene expression of UGT1A1 and MRP2. Cells were cultured for 72 h. Reverse transcription polymerase chain reaction–examined gene expression levels of UGT1A1 and MRP2. (B) Immunofluorescence staining of MRP2. Cells were cultured for 72 h and then fixed and stained for MRP2 (in green). The cell nuclei were stained with DAPI (in blue). Scale bar, 40 μm. (C) MRP2 transporter activity assay. CDF were accumulated in the bile canaliculus in HepG2-DP cells (Arrows). Scale bar, 40 μm. (D) Fluorescence intensity of CDF. The fluorescence intensity of CDF (Ex. 488 nm/Em. 517 nm) accumulated in the bile canaliculi was measured. The values are the mean ± standard deviation for five samples. Statistical analysis was performed by applying the t test (*p < 0.05).CC BY · PubMed Central
Figure 5 Hepatic function in differentiated HepG2-DP cells. (A) Phase-construct image of HepD2-DP cells cultured in differentiation medium. The cells were cultured in the differentiation medium for 0, 7, and 14 days. Scale bar, 100 μm. (B) Gene expression of CYPs, ALB, AFP, bile acid conjugation enzymes, and bile acid transporters in HepG2-DP cells. Cells were cultured for 72 h. Reverse transcription polymerase chain reaction–examined gene expression levels of CYP1A2, CYP3A4, ALB, AFP, UGT1A1, UGT1A3, BSEP, and MRP2. Diff (-) indicates the control and Diff ( +) indicates the differentiation condition. (C) Albumin production of differentiated cells. Albumin production per 24 h was quantified by enzyme-linked immunosorbent assay. D) CYP1A2 and CYP3A4 activity in differentiated cells. HepG2-DP cells were cultured in the differentiation medium for 14 days. HepG2-WT and PHHs were cultured for 48 h and then treated with the indicated inducers for 24 h. CYPs activities were examined using the P450-Glo assay kit. White bars indicate the no CYP inducer condition and black bars indicate the CYP inducer condition. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the t test (*p < 0.05).
Figure 5 Hepatic function in differentiated HepG2-DP cells. (A) Phase-construct image of HepD2-DP cells cultured in differentiation medium. The cells were cultured in the differentiation medium for 0, 7, and 14 days. Scale bar, 100 μm. (B) Gene expression of CYPs, ALB, AFP, bile acid conjugation enzymes, and bile acid transporters in HepG2-DP cells. Cells were cultured for 72 h. Reverse transcription polymerase chain reaction–examined gene expression levels of CYP1A2, CYP3A4, ALB, AFP, UGT1A1, UGT1A3, BSEP, and MRP2. Diff (-) indicates the control and Diff ( +) indicates the differentiation condition. (C) Albumin production of differentiated cells. Albumin production per 24 h was quantified by enzyme-linked immunosorbent assay. D) CYP1A2 and CYP3A4 activity in differentiated cells. HepG2-DP cells were cultured in the differentiation medium for 14 days. HepG2-WT and PHHs were cultured for 48 h and then treated with the indicated inducers for 24 h. CYPs activities were examined using the P450-Glo assay kit. White bars indicate the no CYP inducer condition and black bars indicate the CYP inducer condition. The values are the mean ± standard deviation for three samples. Statistical analysis was performed by applying the t test (*p < 0.05).CC BY · PubMed Central

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Metadata

Authors
Tanaka-yachi, R., Aizawa, K., Shimizu, K., Akutsu, H., & Nakamura, K.
Affiliations
National Research Institute for Child Health and Development (JP)
Corresponding author
Nakamura, Kazuaki
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2022-12-07
Volume
12
Issue
1
Pages
21173-21173
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-022-25777-z
PMC
PMC9729623
OpenAlex
W4311581076
Cited by
4
References
33
Citations by year
2026: 2 · 2025: 2
FWCI
0.30
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Japan Agency for Medical Research and Development (JP21be0304501)
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
Keywords
HepG2 cells, DNMT1, PKR, hepatocyte differentiation, zebularine, DNA methylation inhibition, liver function, CYP1A2, drug metabolism, transcription factors, HNF4α, HNF1B, nuclear receptors, lipid droplets, hepatocyte cell lines
MeSH terms
Cell Differentiation, Humans

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Tanaka-yachi, R., Aizawa, K., Shimizu, K., Akutsu, H., & Nakamura, K. (2022). DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability. Scientific Reports. https://doi.org/10.1038/s41598-022-25777-z