Xuemei Chen Ying Liu Qiujing Zhou Chenxi Zhang Wei Wang Menglong Xu Yaqiang Zhao Wenfeng Zhao Dian Gu Shuhua Tan ^*

• School of Life Science and Technology, China Pharmaceutical University, Nanjing, China

Background: 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. Methods: By in silico analyses, miR-99a-5p was predicted to bind to human PCSK9 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. Results: 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 up-regulates 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 PCSK9 mRNA via a luciferase reporter assay in combination with site-directed mutagenesis. Conclusions: MiR-99a-5p potently down-regulates the expression of PCSK9 by directly interacting with a target site in the human PCSK9 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.