Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Biochem Pharmacol. 2010 Apr 1;79(7):1045-52. doi: 10.1016/j.bcp.2009.11.015. Epub 2009 Nov 27.
Human CYP2E1 is one of the pharmacologically and toxicologically important cytochrome P450 isoforms. Earlier studies have reported that the CYP2E1 expression is extensively regulated by post-transcriptional and post-translational mechanisms, but the molecular basis remains unclear. In the present study, we examined the possibility that microRNA may be involved in the post-transcriptional regulation of human CYP2E1. In silico analysis identified a potential recognition element of miR-378 (MRE378) in the 3'-untranslated region (UTR) of human CYP2E1 mRNA. Luciferase assays using HEK293 cells revealed that the reporter activity of the plasmid containing the MRE378 was decreased by co-transfection of precursor miR-378, indicating that miR-378 functionally recognized the MRE378. We established two HEK293 cell lines stably expressing human CYP2E1 including or excluding 3'-UTR. When the precursor miR-378 was transfected into the cells expressing human CYP2E1 including 3'-UTR, the CYP2E1 protein level and chlorzoxazone 6-hydroxylase activity were significantly decreased, but were not in the cells expressing CYP2E1 excluding 3'-UTR. In both cell lines, the CYP2E1 mRNA levels were decreased by overexpression of miR-378, but miR-378 did not affect the stability of CYP2E1 mRNA. In a panel of 25 human livers, no positive correlation was observed between the CYP2E1 protein and CYP2E1 mRNA levels, supporting the post-transcriptional regulation. Interestingly, the miR-378 levels were inversely correlated with the CYP2E1 protein levels and the translational efficiency of CYP2E1. In conclusion, we found that human CYP2E1 expression is regulated by miR-378, mainly via translational repression. This study could provide new insight into the unsolved mechanism of the post-transcriptional regulation of CYP2E1.
人细胞色素 P4502E1（CYP2E1）是药理学和毒理学上重要的细胞色素 P450 同工酶之一。早期的研究报道称，CYP2E1 的表达受到转录后和翻译后机制的广泛调控，但分子基础尚不清楚。本研究探讨了 microRNA 可能参与人 CYP2E1 转录后调控的可能性。通过计算机分析，在人 CYP2E1 mRNA 的 3'-非翻译区（UTR）中鉴定出 microRNA-378（miR-378）的潜在识别元件（MRE378）。使用 HEK293 细胞进行的荧光素酶测定显示，包含 MRE378 的质粒的报告基因活性在共转染前体 miR-378 时降低，表明 miR-378 可对 MRE378 进行功能性识别。我们建立了稳定表达人 CYP2E1 的两种 HEK293 细胞系，包括或不包括 3'-UTR。当含有 3'-UTR 的细胞中转入前体 miR-378 时，CYP2E1 蛋白水平和氯唑沙宗 6-羟化酶活性显著降低，但在不包含 3'-UTR 的细胞中则没有。在两种细胞系中，miR-378 的过表达均降低了 CYP2E1 mRNA 水平，但 miR-378 不影响 CYP2E1 mRNA 的稳定性。在 25 个人肝组织样本中，CYP2E1 蛋白与 CYP2E1 mRNA 水平之间未观察到正相关，支持转录后调控。有趣的是，miR-378 水平与 CYP2E1 蛋白水平和 CYP2E1 的翻译效率呈负相关。总之，我们发现人 CYP2E1 的表达受 miR-378 调节，主要通过翻译抑制。本研究可为 CYP2E1 转录后调控的未解之谜提供新的见解。
