Construction And Identification Of RNAi Lentivirial Vector Targeting Human LOX-1Gene And Its Effect On The Apoptosis Of HUVECs

Objective1、 Construct RNA interference (RNAi) lentivirial vectors targeting human lectin-like oxidized low density lipoprotein receptor-1(LOX-1) gene and prepare mature virus, and screening the best target sequence.2、Research the effects on the apoptosis of the human umbilical vein endothelial cells(HUVECs) induced by oxidized low density lipoprotein(ox-LDL) with the silencing expression of LOX-1gene.Methods1、Three target sequences of LOX-1gene(OLR1) were designed. DNA oligo nucleotides of target sequence 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-1was co-transfected into293T cells with packaging plasmid by lipofectamine2000.Virus in the supernatant was collected and the virus titer was measured.2、Under the best multiplicity of infection(MOI) and transfection conditions, HUVECs were transfected and divided into control group,negative control group,and LV-OLR1-RNAi-1,LV-OLR1-RNAi-2,LV-OLR1-RNAi-3groups.96h after transfection,the expression of mRNA and protein of LOX-1were respectively detected by RT-PCR and Western Blotting. 3、HUVECs were transfection with the best target sequence to inhibit the expression of LOX-1gene.72h after transfection.HUVECs were cultured with ox-LDL,which the final concentration was150μg/ml.24h after cultured by ox-LDL,cell vitality and proliferation of HUVECs with LOX-1-RNAi was determined by MTT assay.The apoptosis of endothelial cells was assayed by flow cytometry.At the same time,the changes in the expression level of apoptosis-related proteins Bcl-2/Bax were detected by Western Blotting. Results1、PCR and DNA sequencing demonstrated that the inserted sequences were correct.The virus titer were (4-6)×108TU/ml.2、By observing the expression of green fluorescent protein(GFP),When MOI was20and the best transfection conditions were complete medium+5∑g/mlPolybrene,the transfection efficiency was above90%,and HUVECs were at a good growth state.After96h of transfection.the expression of GFP was continuous and stable.3、The results of RT-PCR and Westen Blotting showed that there were not statistical significance on the expression of mRNA and protein of LOX-1between control group and negative group(P>0.05);there were statistical significance between the three transfection groups of LOX-1-RNAi and control group and negative group(P<0.05),also were statistical significance in the transfection groups of LOX-1-RNAi;OLR1-RNAi-3group has the best inhibition efficiency.4、The results of MTT assay indicated that with ox-LDL treatment,the cell vitality of the group without intervention of LOX-1expression was lower than the vitality of control group,and cell proliferation was inhibited,these differences had statistical significance (P<0.05);at the same time HUVECs pretreated with the transfection of LV-OLR1-RNAi to inhibit the expression of LOX-1,the cell vitality was higher than the group without intervention of LOX-1,and cell proliferation increased,the differences had statistical significance (P<0.05);flow cytometry results also indicated that with the inhibition of LOX-1,apoptosis rate of endothelial cells which induced by ox-LDL was significantly lower than the group without transfection (P<0.05).5、The results of Western Blotting showed that ox-LDL increased the expression of apoptosis proteins Bcl-2was down-regulated after ox-LDL treatment,wherease the level of anti-apoptosis proteins Bax was significantly up-regulated,and Bcl-2/Bax ratio decreased;at the same time the Bcl-2/Bax ratio was significantly increaser in HUVECs pretreated with the transfection of LV-OLR1-RNAi group compared with ox-LDL cultured group,and the differences had statistical significance (P<0.05). Conclusion1、RNAi lentivirial vectors targeting LOX-1were constructed successfully.The expression of LOX-1can be inhibited effectively in HUVECs.2、The inhibition of LOX-1can protect endothelial cells against ox-LDL-induced apoptosis,by the way of down-regulating the expression of Bax,up-regulating the expression of Bcl-2and increasing the Bcl-2/Bax ratio,which played the role of anti-atherosclerosis.