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Fig. 3 | BMC Medical Genomics

Fig. 3

From: Integrative analysis of loss-of-function variants in clinical and genomic data reveals novel genes associated with cardiovascular traits

Fig. 3

In vitro validation of candidate genes for lowering plasma cholesterol and triglycerides. a Schematic illustration of lipoprotein metabolism in vivo, and b An in vitro HepG2 cell model to validate three hepatic plasma cholesterol and three hepatic triglyceride candidate genes. I) Cholesterol- and triglyceride-containing very low density lipoprotein (VLDL) particles are synthesized in, and secreted from, the liver to circulation elevating plasma levels of cholesterol and triglycerides. II) The VLDL particles then travel in blood to microcirculation in peripheral tissues, such as the skeletal muscle and adipose tissue, where lipoprotein lipase (LPL) anchored to the endothelium mediates hydrolysis of VLDL-triglycerides forming free fatty acids that are taken up by the local tissue. This extra-hepatic process lowers plasma triglyceride levels. III) The LPL-mediated hydrolysis of VLDL particles results in the formation of smaller cholesterol-rich low-density lipoproteins (LDL) particles. Some LDL particles are taken up by LDL receptors in extra-hepatic tissues, a process that lowers plasma levels of LDL and cholesterol. IV) The most important regulatory process of plasma LDL and cholesterol levels is, however, the uptake of LDL by hepatic LDL receptors. V) Last, uptake of LDL by the LDL receptor in the liver is inhibited by hepatic synthesis of PCSK9 that binds to the LDL receptors and permits their recirculation away from the hepatocyte cell surface (and LDL receptor degradation) effectively lowering the uptake of LDL particles. Thus, high levels of hepatic PCKS9 lead to reduced LDL uptake and higher plasma cholesterol levels. The HepG2 in vitro model of the liver was chosen as it is the most important organ to control plasma triglyceride and cholesterol levels (a) and since all lipid-associated candidate genes were identified in STARNET liver RNA-seq data. However as illustrated in panels a and b, an in vitro model of the liver in the form of HepG2 cells cannot fully model lipid metabolism and the extrahepatic tissue contribution in vivo. c For the plasma cholesterol-lowering candidate genes (RNMTL1, SCRN2 and PCK2), Apolipoprotein B-100 (APOB-100), Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) protein levels were measured in the cell media whereas LDL-receptor (LDLR) was measured in cell lysates. d For the plasma triglyceride-lowering candidate genes (APOC3, SLC39A5, NMRAL1, ABHD14B), Apolipoprotein B-100 (APOB-100), triglycerides (TG) were measured in cell media and lysates. In all experiments, the silencing efficiency of each gene resulted in more than 90% decrease in gene expression measured after 72 h incubation, including 24 h treatment with oleic acid performed before cell harvesting. Values are means ± SEM. Results are based on 3 biological replicates. p < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***). IDL, intermediate-density lipoprotein. Computational analysis results of the genes tested in (c) and (d) are shown in Additional file 1: Figure S9

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