Effects Of PCSK9 On HepG2 Cell Functions And Generation Of D374Y Mutant Transgenic Pigs

PCSK9 is the main gene of human hypercholesterolemia. It involves in meditating intracellular lysosomal degradation pathway of liver cell LDLR, which inhibits scavenging effect of LDL-c in liver, so that the circulation cholesterol level increases. The high expression of PCSK9 or gain of function mutations has a direct relationship with atherosclerotic disease, but also affect the liver function, repair of liver injury, cholesterol transport properties. In order to assess the potential effect of PCSK9 overexpression on liver cell function, we used Hep G2 cells to detect phenotypes such as proliferation, migration, cholesterol transport. Results showed that, overexpression PCSK9 protein in Hep G2 cells did not affect the proliferative capacity, but significantly inhibited the ability of cell migration and cholesterol efflux. Suppression effect of PCSK9 on liver cell migration had close relationship with the regulation of cholesterol. Random distribution of free cholesterol in the cell membrane interfered the activity of small G protein and the conduction of signal pathway involved in lipid raft associated receptors. It also participated in the Erk signaling pathway. Miniature pigs are an ideal animal model for cardiovascular diseases. However the utility of pigs for diseases model preparation is relatively infrequent because of their individual differences. Inbred lines increased the consistency of the genetic background of the experimental animals, thus consequently, would simplify disease progression of the animal model. In this study, we over-expressed the gain-of-function D374 Y mutation of the human proprotein convertase subtilisin/kexin type 9(PCSK9) gene(the major disease gene of human hypercholesterolemia) in Wuzhishan inbred miniature pigs to simulate pathological arterial changes. The PCSK9 gene driven by APOE promoter was successfully expressed in Hep G2 cells, then transgenic pigs were subsequently obtained by SCNT. The RNA and protein levels demonstrated that the human PCSK9 gene was over-expressed in liver of transgenic pigs. In addition, the histochemical results showed that the expression levels of the low-density lipoprotein receptor(LDLR) in the liver of transgenic pigs were extremely significantly lower than those in wild-type pigs, also obvious liver lesions were observed in the transgenic pigs. This study obtained cloned Wuzhishan inbred miniature pigs over-expressing human PCSK9, which might effectively control the influences caused by individual differences in disease studies and provide an ideal disease animal model for studying hypercholesterolemia.