Abstract

1. miR-199a-5p is an important regulator of many biological processes. However, whether and how CYP enzymes are regulated by miR-199a-5p are unknown. Here, we aimed to investigate the potential role of mmu-miR-199a-5p in regulating CYP2 enzymes.

2. Regulatory effects of mmu-miR-199a-5p on CYP expression were assessed in mouse AML-12 hepatocytes. The metabolic activity of CYP2B10 was probed using cyclophosphamide (CPA) as a specific substrate. The regulatory mechanism was investigated using combined luciferase reporter assays and chromatin immunoprecipitation.

3. Of several important drug-metabolizing CYPs, mmu-miR-199a-5p significantly increased the mRNA levels of Cyp2a10, Cyp2c29, and Cyp2j5 in AML-12 cells with Cyp2a10 altered the most. Consistently, mmu-miR-199a-5p enhanced the expression of CYP2B10 protein and cellular metabolism of CPA. Based on database analysis, Cyp2b10 was not a direct target gene of mmu-miR-199a-5p. Thus, a mediator is necessary for the miRNA regulation of CYP2B10. We found that E4BP4 repressed Cyp2b10 transcription and expression through specific binding to a D-box element in the gene promoter. Moreover, mmu-miR-199a-5p inhibited the expression of E4bp4 at the posttranscriptional level by directly targeting the 59–65 nt segment in its 3′-UTR.

4. In conclusion, mmu-miR-199a-5p positively regulates CYP2B10 expression through inhibiting its repressor E4BP4. Our findings may provide an increased understanding of the complex regulatory pathways for CYP2B10.

摘要

1. miR-199a-5p 是许多生物过程的重要调节因子。然而，CYP 酶是否以及如何被 miR-199a-5p 调节是未知的。在这里，我们旨在研究 mmu-miR-199a-5p 在调节 CYP2 酶中的潜在作用。

2. 在小鼠 AML-12 肝细胞中评估了 mmu-miR-199a-5p 对 CYP 表达的调节作用。CYP2B10 的代谢活性使用环磷酰胺 (CPA) 作为特异性底物进行探测。使用组合的荧光素酶报告基因测定和染色质免疫沉淀研究调节机制。

3. 在几种重要的药物代谢 CYP 中，mmu-miR-199a-5p 显着增加了AML-12 细胞中Cyp2a10、Cyp2c29和Cyp2j5的 mRNA 水平，其中Cyp2a10的变化最大。一致地，mmu-miR-199a-5p 增强了 CYP2B10 蛋白的表达和 CPA 的细胞代谢。根据数据库分析，Cyp2b10不是mmu-miR-199a-5p的直接靶基因。因此，CYP2B10 的 miRNA 调节需要介质。我们发现 E4BP4 通过与基因启动子中的 D-box 元件特异性结合来抑制Cyp2b10 的转录和表达。此外，mmu-miR-199a-5p 抑制E4bp4的表达在转录后水平通过直接靶向其 3'-UTR 中的 59-65 nt 片段。

4. 总之，mmu-miR-199a-5p 通过抑制其阻遏物 E4BP4 正向调节 CYP2B10 的表达。我们的研究结果可能会增加对 CYP2B10 复杂调节途径的理解。