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The Wnt/β-catenin pathway is involved in 2,5-hexanedione-induced ovarian granulosa cell cycle arrest
Xueming Xu1, Yimei Pan1, Liqin Zhan1
1Department of Preventive Medicine, Fujian Provincial Key Laboratory of Environmental Factors and Cancer, Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Fuzhou 350122, Fujian Province, China.
Abstract:
N-Hexane causes significant ovarian toxicity, and its main active metabolite 2,5-hexanedione (2,5-HD) can induce ovarian injury through mechanisms such as inducing apoptosis in ovarian granulosa cells (GCs); however, the specific mechanism has not been fully elucidated. In this study, we investigated the effects on the cell cycle of rat ovarian GCs exposed in vitro to different concentrations of 2,5-HD (0 mM, 20 mM, 40 mM, and 60 mM) and further explored the mechanism by mRNA and miRNA microarray analyses. The flow cytometry results sindicated that compared with control cells, in ovarian GCs, there was significant cell cycle arrest after 2,5-HD treatment. Cell cycle- and apoptosis- related gene (Cdk2, Ccnd1, Bax, Bcl-2, Caspase3, and Caspase9) expression was altered. The mRNA and miRNA microarray results suggested that 5678 mRNAs and 32 miRNAs were differentially expressed in the 2,5-HD-treated group. A total of 262 target mRNAs were obtained by miRNA and mRNA coexpression analysis, forming 368 miRNA-mRNA coexpression relationship pairs with 27 miRNAs. GO and KEGG analyses showed that differentially expressed genes were significantly enriched in the cell cycle and Wnt signaling pathways. Furthermore, significant changes in the expression of Wnt signaling pathway and cell cycle- related genes (Fzd1, Lrp6, Tcf3, Tcf4, Fzd6, Lrp5, β-catenin, Lef1, GSK3β, and Dvl3) after 2,5-HD treatment were confirmed by qRT-PCR and Western blotting. Ther results of dual-luciferase assays indicated decreased β-catenin/TCF transcriptional activity after 2,5-HD treatment. In addition, Wnt pathway-related miRNAs (rno-miR-145-5p, rno-miR-143-3p, rno-miR-214-3p, rno-miR-138-5p, and rno-miR-199a-3p) were changed significantly after 2,5-HD treatment. In summary, 2,5-HD induced cell cycle arrest in ovarian GCs, and the Wnt/β-catenin signaling pathway may play a very critical role in this process. Alterations in the expression of miRNAs such as rno-miR-145-5p may have significant implications.
Insights
N-Hexane metabolite 2,5-hexanedione (2,5-HD) causes ovarian granulosa cell cycle arrest. The Wnt/β-catenin signaling pathway and specific microRNAs are implicated in this toxicity, revealing potential mechanisms of ovarian injury.
Area of Science:
- Toxicology
- Cell Biology
- Molecular Biology
Background:
- N-Hexane induces ovarian toxicity, with 2,5-hexanedione (2,5-HD) as a key metabolite.
- The precise mechanisms of 2,5-HD-induced ovarian granulosa cell (GC) injury, including apoptosis and cell cycle disruption, require further elucidation.
Purpose of the Study:
- To investigate the impact of 2,5-HD on the cell cycle of rat ovarian GCs in vitro.
- To explore the underlying molecular mechanisms, including gene and microRNA expression profiles, and the involvement of signaling pathways.
Main Methods:
- Rat ovarian GCs were exposed to varying concentrations of 2,5-HD.
- Flow cytometry was used to assess cell cycle distribution.
- mRNA and miRNA microarray analyses were performed.
- Quantitative real-time PCR (qRT-PCR) and Western blotting validated gene expression.
- Dual-luciferase assays examined pathway activity.
Main Results:
- 2,5-HD treatment resulted in significant cell cycle arrest in ovarian GCs.
- Expression of cell cycle and apoptosis-related genes (e.g., Cdk2, Bax, Bcl-2) was altered.
- Microarray analysis revealed differential expression of 5678 mRNAs and 32 miRNAs.
- The Wnt/β-catenin signaling pathway and cell cycle pathways were significantly enriched.
- Downregulation of β-catenin/TCF transcriptional activity and altered expression of Wnt pathway-related miRNAs were observed.
Conclusions:
- 2,5-HD induces cell cycle arrest in ovarian GCs.
- The Wnt/β-catenin signaling pathway plays a critical role in 2,5-HD-induced ovarian toxicity.
- Altered miRNA expression, such as rno-miR-145-5p, may contribute to the observed effects.
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