MiR-224-5p Regulates Lipid Uptake By Repressing PCSK9 Expression In HepG2 Cell

Hyperlipidemia is a major risk factor for atherosclerotic cardiovascular disease.Hyperlipidemia contributes to the development of atherosclerosis,and increases atherosclerotic plaque instability.The atheromatous plaque will evoke the form of thrombi,which blocks blood flow,causing coronary heart disease and ischemic stroke.Notably,circulating low-density lipoprotein cholesterol(LDL-C)plays a key role in the initiation and development of atherosclerosis.Lower serum LDL-C reduces atherosclerotic cardiovascular disease events.Proprotein convertase subtilisin/Kexin type 9(PCSK9)can effectively bind to the LDLR on the surface of hepatocytes.PCSK9-LDLR complex is transported to the lysosome compartment and degraded,leading to prevent LDLR-mediated LDL-C clearance.PCSK9 inhibitors are deemed to be a new generation of lipid-lowering drugs following statin.Micro RNAs(miRNAs)are small non-coding RNAs that post-transcriptionally inhibit gene expression usually by binding to the 3′ untranslated regions(3′UTR)of target m RNAs in mammals.miRNAs areclosely related to normal metabolism and occurrence of disease.miRNAs play important roles in the regulation of lipid metabolism,which are considered as new potential therapeutic targets for hyperlipidemia.Objectives: To investigate the role of miR-224-5p in regulating PCSK9 and to observe the effect of miR-224-5p on lipid uptake ability in Hep G2 cells.Methods: First,we analyze location,conservation and seed sequences of the miR-224-5p gene by using the principles and methods of bioinformatics.Next,we analyze binding sites and free energy scores between miR-224-5p and PCSK9 in databases such as Targetscan,miRanda,miRDB,RNAhybrid.Dual–Luciferase Reporter Gene Assay verify that miR-224-5p directly target PCSK9 3′UTR.In addition,Western blot detects PCSK9 and LDLR protein after transfecting miR-224-5p mimic or miR-224-5p inhibitor.Using Cell immunofluorescence to observe the LDLR on cells surface.Oil red O staining and Di I-LDL are utilized to observe the effect of miR-224-5p on lipid droplets accumulation and lipid uptake ability in Hep G2 cells.Results: Bioinformatics analysis revealed that human miR-224-5p gene is located in Xq28.miR-224-5p is broadly conserved among diverse species.The free energy scores for the hybridization between miR-224-5p and PCSK9 are very low in human.Dual–Luciferase Reporter Gene Assay showed that miR-224-5p mimic strongly repressed PCSK9-WT3′UTR luciferase activity.While the luciferase activity of PCSK9-Mut 3′UTR was not affected by miR-224-5p mimic.These suggest that miR-224-5p directly targets PCSK9 3′UTR.Furthermore,we find that miR-224-5p mimic significantly repressed the expression of PCSK9 protein and increased LDLR protein level in Hep G2 cells;on the contrary,down-regulation of miR-224-5p increased expression of PCSK9 and decreased LDLR level.Also,miR-224-5p up-regulation can also promote LDL-C uptake in Hep G2 cells.Moreover,oil red O staining showed that miR-224-5p mimic significantly reduced lipid droplets accumulation in Hep G2 cells;however,miR-224-5p inhibitor increased lipid droplets accumulation.Conclusions: 1.miR-224-5p inhibits the expression of PCSK9 though directly target 3′UTR of PCSK9 m RNA.2.miR-224-5p can reduces content of lipid droplets and promotes lipid uptake in Hep G2 cells.