Abstract
Epigenetic modifications, such as DNA methylation, play an important role in carcinogenesis. In a recent NTP study, chronic exposure of B6C3F1/N mice to Ginkgo biloba extract (GBE) resulted in a high incidence of hepatocellular carcinomas (HCC). Genome-wide promoter methylation profiling on GBE-exposed HCC (2000 mg/kg group), spontaneous HCC (vehicle-control group), and age-matched vehicle control liver was performed to identify differentially methylated genes in GBE-exposed HCC and spontaneous HCC. DNA methylation alterations were correlated to the corresponding global gene expression changes. Compared to control liver, 1296 gene promoters (719 hypermethylated, 577 hypomethylated) in GBE-exposed HCC and 738 (427 hypermethylated, 311 hypomethylated) gene promoters in spontaneous HCC were significantly differentially methylated, suggesting an impact of methylation on GBE-exposed HCC. Differential methylation of promoter regions in relevant cancer genes (cMyc, Spry2, Dusp5) and their corresponding differential gene expression was validated by quantitative pyrosequencing and qRT-PCR, respectively. In conclusion, we have identified differentially methylated promoter regions of relevant cancer genes altered in GBE-exposed HCC compared to spontaneous HCC. Further study of unique sets of differentially methylated genes in chemical-exposed mouse HCC could potentially be used to differentiate treatment-related tumors from spontaneous-tumors in cancer bioassays and provide additional understanding of the underlying epigenetic mechanisms of chemical carcinogenesis.
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Abbreviations
- GBE:
-
Ginkgo biloba extract
- HCC:
-
Hepatocellular carcinoma
- SPNT:
-
Spontaneous
- CNTL:
-
Control
- Myc:
-
Myelocytomatosis
- Spry2:
-
Sprouty RTK signaling antagonist 2
- Dusp5:
-
Dual specificity phosphatase 5
- DEGs:
-
Differentially expressed genes
- DMR:
-
Differentially methylated regions
- DMGs:
-
Differentially methylated genes
- H&E:
-
Hematoxylin and eosin
- FFPE:
-
Formalin-fixed paraffin embedded
- RMA:
-
Robust multiarray normalization
- ANOVA:
-
Analysis of variance
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Acknowledgements
We would like to thank Florida State University Genomics Core and the NIEHS Microarray Core for running the Nimblegen methylation Arrays and Affymetrix Arrays, respectively. We would also like to acknowledge the staff in the NIEHS Pathology Support Core Laboratories and the NTP tissue archives for their expertise. We appreciate Drs. Jian-Liang Li and Alex Merrick for critically reviewing this manuscript.
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Kovi, R.C., Bhusari, S., Mav, D. et al. Genome-wide promoter DNA methylation profiling of hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice. Arch Toxicol 93, 2219–2235 (2019). https://doi.org/10.1007/s00204-019-02505-7
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DOI: https://doi.org/10.1007/s00204-019-02505-7