Chen Xuemei, Liu Ying, Zhou Qiujing, Zhang Chenxi, Wang Wei, Xu Menglong, Zhao Yaqiang, Zhao Wenfeng, Gu Dian, Tan Shuhua
Department of Cell and Molecular Biology, School of Life Science and Technology, State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, China Pharmaceutical University, Nanjing, Jiangsu, China.
Front Genet. 2024 Nov 19;15:1469094. doi: 10.3389/fgene.2024.1469094. eCollection 2024.
MicroRNAs (miRs/miRNAs) play pivotal roles in modulating cholesterol homeostasis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipoprotein receptor (LDLR) at the surface of hepatocytes and accelerates its degradation in lysosomes, thereby impairing the clearance of circulating low-density lipoprotein cholesterol (LDL-C) from plasma. Thus, suppressing PCSK9 expression level has become an effective approach for treating hypercholesterolemia. Here, we sought to identify novel miRNAs that inhibit PCSK9 expression.
By analyses, miR-99a-5p was predicted to bind to human mRNA. Following transfection of miR-99a-5p or anti-miR-99a-5p in human and mouse hepatocytes, qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, LDL-C uptake, and cellular cholesterol measurement were performed.
miR-99a-5p overexpression potently inhibited PCSK9 expression, thereby up-regulating LDLR, functionally enhancing LDL-C uptake and increasing intracellular cholesterol levels in human, but not in mouse, cells. Conversely, anti-miR-99a-5p upregulates PCSK9, leading to a reduction in LDLR, attenuation of LDL-C uptake, and a decrease in the intracellular cholesterol levels of human hepatocytes. Furthermore, miR-99a-5p was shown to bind to the predicted target site "UACGGGU" in the 3'-UTR of human mRNA via a luciferase reporter assay in combination with site-directed mutagenesis.
MiR-99a-5p potently downregulates the expression of PCSK9 by directly interacting with a target site in the human 3'-UTR, thereby up-regulating LDLR and functionally enhancing LDL-C uptake in human hepatocytes. MiR-99a-5p could serve as an inhibitor of PCSK9 for treating hypercholesterolemia to inhibit atherosclerosis.
微小RNA（miR/miRNA）在调节胆固醇稳态中起关键作用。前蛋白转化酶枯草溶菌素9型（PCSK9）与肝细胞表面的低密度脂蛋白受体（LDLR）结合，并加速其在溶酶体中的降解，从而损害血浆中循环低密度脂蛋白胆固醇（LDL-C）的清除。因此，抑制PCSK9表达水平已成为治疗高胆固醇血症的有效方法。在此，我们试图鉴定抑制PCSK9表达的新型miRNA。
通过分析，预测miR-99a-5p与人PCSK9 mRNA结合。在人和小鼠肝细胞中转染miR-99a-5p或抗miR-99a-5p后，进行qRT-PCR、蛋白质印迹、免疫荧光、酶联免疫吸附测定、流式细胞术、LDL-C摄取和细胞胆固醇测量。
miR-99a-5p过表达有效抑制PCSK9表达，从而上调LDLR，在功能上增强人而非小鼠细胞中LDL-C的摄取并增加细胞内胆固醇水平。相反，抗miR-99a-5p上调PCSK9，导致LDLR减少，LDL-C摄取减弱，以及人肝细胞内胆固醇水平降低。此外，通过荧光素酶报告基因测定结合定点诱变，显示miR-99a-5p与人PCSK9 mRNA的3'-UTR中的预测靶位点“UACGGGU”结合。
MiR-99a-5p通过与人PCSK9 3'-UTR中的靶位点直接相互作用，有效下调PCSK9的表达，从而上调LDLR并在功能上增强人肝细胞中LDL-C的摄取。MiR-99a-5p可作为PCSK9的抑制剂用于治疗高胆固醇血症以抑制动脉粥样硬化。
