Jiang Huajun, Zhang Jin, Du Yu, Jia Xiaojian, Yang Fan, Si Shuyi, Wang Li, Hong Bin
Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Tiantan Xili, Beijing 100050, China.
Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Tiantan Xili, Beijing 100050, China.
Atherosclerosis. 2015 Dec;243(2):523-32. doi: 10.1016/j.atherosclerosis.2015.10.026. Epub 2015 Oct 23.
Low-density lipoprotein receptor (LDLR) mediates endocytosis of LDL particles and is important in maintaining plasma cholesterol levels, thus its expression is under extensive regulation at multiple levels, including transcriptional and posttranscriptional regulation by transcription factors (TFs) and RNA-binding proteins (RBPs). Here, we identified microRNA-185 (miR-185) as a novel direct posttranscriptional regulator of LDLR and an indirect LDLR modulator through KSRP in hepatic cells.
Using quantitative real-time PCR (qPCR), we detected the effect of predicted LDLR-targeting miRNAs and found that overexpression of miR-185 repressed LDLR expression and LDL uptake in HepG2 cells by 62.4 ± 6.0% (p = 7.0 × 10(-5)) and 32.5 ± 6.0% (p = 7.7 × 10(-4)) respectively, through directly targeting LDLR 3'UTR. Unexpectedly, the antisense inhibitor of miR-185 had similar repression effect on LDLR although it reduced the association of endogenous miR-185 with LDLR mRNA. Further experiments revealed that KH-type splicing regulatory protein (KSRP), one of the LDLR-destabilizing RBPs, is also a target of miR-185. KSRP silencing reversed the repression effects of miR-185-inhibitor on LDLR. Thus miR-185 regulates LDLR expression not only through directly targeting but also by a RBP-involved indirect pathway. Finally, the in vivo results showed that miR-185-inhibitor upregulated hepatic LDLR expression and correlated with a decrease in plasma cholesterol level and arterial plaque area in ApoE KO mice.
These findings reveal that miR-185 controls cholesterol homeostasis as a key posttranscriptional LDLR modulator in hepatic cells, providing novel insight into the regulatory mechanism for LDLR expression and the anti-atherosclerosis effect of miR-185-inhibitor.
低密度脂蛋白受体（LDLR）介导低密度脂蛋白颗粒的内吞作用，对维持血浆胆固醇水平至关重要，因此其表达在多个水平受到广泛调控，包括转录因子（TFs）和RNA结合蛋白（RBPs）的转录及转录后调控。在此，我们鉴定出微小RNA-185（miR-185）是LDLR一种新的直接转录后调节因子，且在肝细胞中通过KSRP间接调节LDLR。
使用定量实时PCR（qPCR），我们检测了预测靶向LDLR的微小RNA的作用，发现miR-185过表达分别通过直接靶向LDLR 3'UTR使HepG2细胞中LDLR表达和LDL摄取分别降低62.4±6.0%（p = 7.0×10⁻⁵）和32.5±6.0%（p = 7.7×10⁻⁴）。出乎意料的是，miR-185的反义抑制剂对LDLR有类似的抑制作用，尽管它降低了内源性miR-185与LDLR mRNA的结合。进一步实验表明，KH型剪接调节蛋白（KSRP）是一种使LDLR不稳定的RBP，也是miR-185的一个靶点。KSRP沉默逆转了miR-185抑制剂对LDLR的抑制作用。因此，miR-185不仅通过直接靶向而且通过涉及RBP的间接途径调节LDLR表达。最后，体内实验结果表明，miR-185抑制剂上调了ApoE基因敲除小鼠肝脏LDLR表达，并与血浆胆固醇水平降低和动脉斑块面积减小相关。
这些发现揭示，miR-185作为肝细胞中LDLR关键的转录后调节因子控制胆固醇稳态，为LDLR表达的调控机制及miR-185抑制剂的抗动脉粥样硬化作用提供了新见解。
