Work

MiR-99a-5p up-regulates LDLR and functionally enhances LDL-C uptake via suppressing PCSK9 expression in human hepatocytes

Year: 2024

Type: article

Abstract: 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 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 PCSK9 mRNA via a luciferase reporter assay in combination with site-directed mutagenesis. Conclusion MiR-99a-5p potently downregulates 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.

Source: Frontiers in Genetics

DOI: 10.3389/fgene.2024.1469094