Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, (X.X., L.M., J.Y., C.F., C.L., M.G., M.L., L.G., X.Q., J.R.), Laboratory of Neural Signal Transduction, Institute of Neuroscience, Chinese Academy of Sciences (Y.W.), Shanghai, China.
Drug Metab Dispos. 2013 Oct;41(10):1732-7. doi: 10.1124/dmd.113.052217. Epub 2013 May 23.
Mouse cytochrome P450 2b9 (Cyp2b9) is a testosterone 16α-hydroxylase enzyme showing female-specific expression in many inbred mouse strains, including C57BL/6J. Previous studies have recognized that some sex-dependently secreted endogenous modulating factors were involved in the sexually dimorphic expression of Cyp2b9 through transcriptional regulation. In this study, we found evidence that some microRNAs contributed to the sexually biased expression of Cyp2b9 via post-transcriptional regulation. Cyp2b9 was upregulated in livers of hepatocyte-specific Dicer1 knockout mice at 3 weeks. The age-dependent downregulation of Cyp2b9 in the livers of male mice was diminished when Dicer1 was specifically knocked out in hepatocytes. When these data were combined with bioinformatics analysis and microRNA profiles of male and female mice, we found that 18 microRNAs were associated with the sexually dimorphic expression of Cyp2b9, which showed higher expression levels in male C57BL/6J mice when compared with females. Luciferase assays revealed that approximate half of these microRNAs repressed luciferase activity in a reporter system containing the 3'-untranslated region (3'-UTR) of Cyp2b9, and also inhibited Cyp2b9 protein expression. MicroRNA seed region mutation or mutations in putative binding sites of the microRNAs in Cyp2b9 3'-UTR led to the loss of the suppression of luciferase activity. There was also a negative correlation between the levels of these microRNAs and Cyp2b9. Our results suggested that multiple microRNAs participated in the regulation of Cyp2b9 expression, and that the lower expression levels of these microRNAs potentially contributed to the female-specific expression of Cyp2b9 in the livers of C57BL/6J mice.
小鼠细胞色素 P450 2b9（Cyp2b9）是一种睾酮 16α-羟化酶，在许多近交系小鼠品系中表现出雌性特异性表达，包括 C57BL/6J。先前的研究已经认识到，一些性别依赖性分泌的内源性调节因子通过转录调节参与 Cyp2b9 的性别二态性表达。在这项研究中，我们发现了一些 microRNAs 通过转录后调控参与 Cyp2b9 性别偏性表达的证据。在 3 周龄时，肝细胞特异性 Dicer1 敲除小鼠肝脏中的 Cyp2b9 上调。当 Dicer1 特异性敲除肝细胞时，雄性小鼠肝脏中 Cyp2b9 的年龄依赖性下调减少。当将这些数据与生物信息学分析和雄性和雌性小鼠的 microRNA 谱相结合时，我们发现 18 个 microRNAs 与 Cyp2b9 的性别二态性表达相关，当与雌性小鼠相比时，这些 microRNAs 在雄性 C57BL/6J 小鼠中表现出更高的表达水平。荧光素酶测定显示，这些 microRNAs 中有一半左右在包含 Cyp2b9 3'-非翻译区（3'-UTR）的报告系统中抑制荧光素酶活性，并抑制 Cyp2b9 蛋白表达。microRNA 种子区突变或 Cyp2b9 3'-UTR 中 microRNA 结合位点的突变导致荧光素酶活性抑制的丧失。这些 microRNAs 的水平与 Cyp2b9 之间也存在负相关。我们的结果表明，多个 microRNAs 参与 Cyp2b9 表达的调节，并且这些 microRNAs 的较低表达水平可能有助于 C57BL/6J 小鼠肝脏中 Cyp2b9 的雌性特异性表达。
