Abstract
Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3′-untranslated regions (3′-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.
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Abbreviations
- AsOs:
-
Antisense oligonucleotides
- CNTL:
-
Control
- DEGs:
-
Differentially expressed genes
- DmiRs:
-
Differentially expressed miRNAs
- FC:
-
Fold change
- FDR:
-
False discovery rate
- FFPE:
-
Formalin-fixed paraffin embedded
- GBE:
-
Ginkgo biloba extract
- H&E:
-
Hematoxylin and eosin
- HCC:
-
Hepatocellular carcinoma
- miRNA:
-
MicroRNA
- mmu-miR:
-
Mus musculus MicroRNA
- MRE:
-
MiRNA response element
- NTP:
-
National Toxicology Program
- RMA:
-
Robust multiarray normalization
- SPNT:
-
Spontaneous
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Acknowledgements
We would like to thank DNTP (ES103319-03) and DIR, NIEHS for funding this project. This work was done while Dr. Haruhiro Yamashita was on a sabbatical at the National Toxicology Program, NIEHS. We would like to thank NIEHS Microarray Core and Cellular and Molecular Pathology Branch, Pathology Support Core for their technical assistance on this project. We would like to express our appreciation for Drs. Alison Harrill and Pierre Bushel for critically reviewing this manuscript.
Funding
This work was supported by the Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC.
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Yamashita, H., Surapureddi, S., Kovi, R.C. et al. Unique microRNA alterations in hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice. Arch Toxicol 94, 2523–2541 (2020). https://doi.org/10.1007/s00204-020-02749-8
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DOI: https://doi.org/10.1007/s00204-020-02749-8