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Construction and Identification of RNAi Lentivirial Vector Targeting Human LOX-1 Gene
Li Jun1, Tao Guizhou1*, Huang Jianhua2
1Department of Cardiology, the First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, China; 2Department of Key Laboratory of Tissue Engineering, the First Affiliated Hospital of Liaoning Medical University, Jinzho
Abstract: This research designed three target sequences (OLR1-RNAi-1， OLR1-RNAi-2， OLR1-RNAi-3) based on LOX-1 gene (OLR1) sequence and shRNA designed principles. DNA oligos of target sequences were synthesized and cloned into lentivirial vector U6-vshRNA-CMV-GFP. The recombined vector was confirmed by PCR and DNA sequencing. Lentiviral vector shRNA-LOX-1 was co-transfected into 293T cells with packaging plasmid by Lipofectamine 2000. Virus in the supematant was collected and the virus titer was measured. Under the best transfection conditions， HUVEC were transfected. The expressions of mRNA and protein of LOX-1 were respectively detected by RT-PCR and Western blot. The apoptosis of HUVEC which were cultured with ox-LDL was assayed by flow cytometry. PCR and DNA sequencing demonstrated that the inserted sequences were correct. The expression of LOX-1 of RNAi transfection groups were inhibited by different degree. With the inhibition of LOX-1， apoptosis rate of endothelial cells which induced by ox-LDL was significantly lower than the group without transfection. RNAi lentivirial vectors targeting LOX-1 were constructed successfully. The expression of LOX-1 can be inhibited effectively in HUVEC， and the inhibition of LOX-1 can protect endothelial cells against ox-LDL-induced apoptosis， which laid the foundation for the further study on the protection mechanism of endothelial cell injury.
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