NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing 100050, China; Beijing Vocational College of Agriculture, No.5 Daotian Nanli, Beijing, 102442, China.
NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, No.1 Tiantan Xili, Beijing 100050, China.
Atherosclerosis. 2022 May;348:56-67. doi: 10.1016/j.atherosclerosis.2022.03.007. Epub 2022 Mar 5.
Cholesterol 7alpha-hydroxylase (CYP7A1) is the rate limiting enzyme of the bile acid biosynthetic pathway to convert cholesterol to bile acids, which is a major output pathway for cholesterol catabolism. In this study, we aimed to assess the potential regulatory mechanisms of microRNA-185 (miR-185) in cholesterol and bile acid homeostasis.
Eight-week-old male ApoE KO mice fed a high-fat diet (HFD) were injected with lentiviruses encoding antisense miR-185 (miR-185-inh). Microarrays were applied to profile miR-185-regulated genes involved in bile acid metabolism. The expression of potential targets of miR-185 was validated using qPCR and Western blotting assay in human hepatoma HepG2 cells.
The administration of miR-185-inh correlated with decreased serum total bile acids levels in ApoE KO mice. Microarray gene profiling revealed that inhibition of miR-185 upregulated hepatic CYP7A1 expression in vivo, which was further validated in HepG2 cells and primary hepatic cells in vitro by overexpression or inhibition of miR-185. Furthermore, it was revealed that miR-185 regulated CYP7A1 expression via a FoxO1-involved indirect pathway and that miR-185 directly modulated FoxO1 expression by binding to its mRNA 3'UTR in a traditional post-transcriptional manner. Besides, we also observed that miR-185 regulated CYP7A1 expression by increasing p-AKT/AKT level, which induced the phosphorylation of FoxO1 and promoted FoxO1 degradation at a post-translational level.
This study provides convincing evidence on the critical role of miR-185 in FoxO1 modulation at both post-transcriptional and post-translational levels, which accounts for the effects on CYP7A1 gene and its mediated cholesterol-bile acid metabolism. These results suggest an important role of miR-185 as a novel atherosclerosis-protective target for drug discovery.
胆固醇 7α-羟化酶（CYP7A1）是胆汁酸生物合成途径中的限速酶，可将胆固醇转化为胆汁酸，这是胆固醇分解代谢的主要输出途径。在这项研究中，我们旨在评估 microRNA-185（miR-185）在胆固醇和胆汁酸稳态中的潜在调节机制。
将编码反义 miR-185 的 lentiviruses 注射到喂食高脂肪饮食（HFD）的 8 周龄雄性 ApoE KO 小鼠中（miR-185-inh）。应用微阵列技术对参与胆汁酸代谢的 miR-185 调控基因进行基因谱分析。在 HepG2 细胞和原代肝细胞中通过 qPCR 和 Western blotting 实验验证 miR-185 的潜在靶基因的表达。
miR-185-inh 的给药与 ApoE KO 小鼠血清总胆汁酸水平降低相关。微阵列基因谱分析显示，体内抑制 miR-185 可上调肝 CYP7A1 表达，在 HepG2 细胞和原代肝细胞中通过过表达或抑制 miR-185 进一步验证。此外，结果表明 miR-185 通过 FoxO1 参与的间接途径调节 CYP7A1 的表达，并且 miR-185 通过与其 mRNA 3'UTR 结合以传统的转录后方式直接调节 FoxO1 的表达。此外，我们还观察到 miR-185 通过增加 p-AKT/AKT 水平调节 CYP7A1 表达，这诱导 FoxO1 的磷酸化，并在翻译后水平促进 FoxO1 的降解。
本研究提供了令人信服的证据，证明 miR-185 在 FoxO1 的转录后和翻译后水平调节中起关键作用，这解释了其对 CYP7A1 基因及其介导的胆固醇-胆汁酸代谢的影响。这些结果表明 miR-185 作为一种新的抗动脉粥样硬化药物发现的靶点具有重要作用。
