Construction of small hair RNA lentiviral vectors targeting PCSK9 gene and its silent effects on PC-SK9 gene

Objective To construct lentiviral vector-based short hairpin RNA(shRNA) targeted at human PCSK9 and evaluate its inhibitory effects on the expression of PCSK9 gene.Methods shRNAs targeted at human PCSK9 RNA were designed and cloned into plentilox 3.7 plasmid.The three recombinant plasmids(shRNA-1,shRNA-2 and shRNA-3)were identified by enzyme digestion and sequencing.Positive plasmids were co-transfeeted with pCDNA3-PCSK9-FLAG to 293 cells to test their knockdown functions using Western blotting methods.Cells transfected with empty plentilox3.7 was used as the control.Recombinant lentivirus was produced by co-transfecting hshRNA-2、pCre-VSV-G and pLoxp-CMV-R8.91 into 293 packaging cells.293 cells were then infected with lentiviral hshRNA-2,lentiviral control shRNA,or empty lentivirus. After 48 hours,the cells were transfected with pCDNA3-hPCSK9-FLAG and the PCSK9 expression was measured by Western blotting methods.Results The shRNA lentiviral vectors targeting PCSK9 gene were confirmed by double enzyme digestion and sequencing.HshRNA-2 showed high inhibitory efficacy. Lentiviral hshRNA-2 decreases the expression of human PCSK9 protein markedly.Conclusions lentiviral vector-based shRNA targeted at human PCSK9 could knock down their gene expressions specifically by RNA interference(RNAi) technology,which provided a useful tool for investigating PCSK9-specific shRNA in regulating lipid metabolism.