The Effect Of SCAP Dysregulation On Cholesterol Metabollism Pathway

Background:We have demonstrated that cytokines can increase cholesterol sensor SCAP expression to disrupt cholesterol-mediated LDLr and HMG-CoAR feedback regulation. The aim of this study was to investigate the effect of SCAP dysregulation (overexpression or gene silence) on cholesterol metabolism pathway in HMCs.Methods:The HMCs were transfected with DNA plasmid or SCAP siRNA by electroporation to overexpress or knockdown SCAP. Intracelluar lipid level of HMCs was assessed by Oil Red O staining and quantitative measurement intracellular cholesterol. Total cellular RNA was isolated from cells for detecting SCAP, LDLr and HMG-CoAR mRNA levels using real-time PCR. nSREBP-2 (N terminal of SREBP2), LDLr, HMG-CoAR and PCSK9 protein expression were examined by western blotting.Results:The transfection was successful by showing that mRNA expression of SCAP was increased or decreased in HMCs after transfection. SCAP overexpression increased intracellular lipid droplets accumulation and cholesterol level. SCAP gene silence decreased intracellular lipid droplets accumulation and cholesterol level under inflammatory stress. Next, we evaluated SREBP-2 translocation by measuring nSREBP-2. The results demonstrated that SCAP o/e increased nSREBP-2, thereby enhancing LDLr and HMG-CoAR mRNA expression accordingly. Moreover, SCAP overexpression disrupts cholesterol-mediated LDLr and HMG-CoAR feedback regulation both on mRNA level and protein level. However, LDLr protein remained unchanged ofter SCAP overexpression, suggesting that there were some post-transcriptional regulations. SCAP gene silence decreased LDLr, HMG-CoAR mRNA expression and intracellular lipid level under inflammatory stress.