Inhibition Of Ang2 Expression In Malignantmelanoma Cells Using RNAi Technology

ObjectiveTo construct and identify the RNAi lentiviral expression vector for Ang2 gene for studying the role of it,s intervention of Ang2 gene of malignant melanoma cells. It would be useful for the further experiment of the inhibition of the growth of nude mouse transplantable tumor, and it would provide experiment evidence for tumor gene therapy.Methods:1,Connect the recombinant plasmids of pSilencer1.0-U6-Ang2-siRNA and pNL-EGFP vectors which were identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI. And the Lentiviral transfer plasmids pNL-EGFP-U6-Ang2-siRNA were constructed, then identified by using enzyme digestion electrophoresis of the restriction endonuclease XbaI and sequencing analysis.2,Lentiviral vector systems of three plasmids[1,2]were constituted using pNL-EGFP-U6-Ang2-Ⅰand pNL-EGFP-U6-Ang2-Ⅱ,pVSVG and pHelper,The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Ang2-Ⅰand pNL-EGFP-U6-Ang2-Ⅱlentivirus.3,Collecting the virus supernatant and using the different density of the collected supernatant to determination the viral titer.4,To choice an appropriate infection plural number for further experiment by using different infection plural number of lentivirus to infect malignant melanoma cells.5,Malignant melanoma cells were infected by lentivirus and Realtime RT-PCR was conducted to examine inhibitory efficiency of the expression of Ang2 gene. Results:1,The U6-Ang2-siRNA was constructed successfully from pSilencer 1.0-U6-Ang2-siRNA by using enzyme digestion electrophoresis of the restriction endonuclease XbaI.2,The pNL-EGFP carrier was cut correctly by using enzyme digestion electrophoresis of the restriction endonuclease XbaI.3,The lentiviral transfer plasmids of pNL-EGFP-U6-Ang2-Ⅰand pNL-EGFP-U6-Ang2-Ⅱwere constructed successfully and identified by using enzyme digestion electrophoresis of XbaI and sequencing analysis. The results fully in line with expectations.4,pNL-EGFP-U6-Ang2-Ⅰ,pNL-EGFP-U6-Ang2-Ⅱvirus were produced by using the three plasmid lentiviral packaging system. The virus supernatant was collected and viral titers measured for the 8.0×103/ul.5,The infection efficiency that pNL-EGFP-U6-Ang2-Ⅰand pNL-EGFP-U6-Ang2-Ⅱlentivirus infected malignant melanoma cells reaches as high as 94.6%.The results of Reatime RT-PCR demonstrated that the pNL-EGFP-U6-Ang2-Ⅰlentivirus could inhibit the expression of Ang2 genes in malignant melanoma cells, and the rate of the inhibition was 68.31%(p<0.05), but the expression of Ang2 genes in malignant melanoma cells trasduced with pNL-EGFP-U6-Ang2-Ⅱlentivirus inhibited is low, the rate was 13.31%,with has no statistical significance(p>0.05).Conclusions:Lentivector carrying Ang2-siRNA was constructed successfully by using the three plasmid lentiviral packaging system, and it was able to inhibit the expression of Ang2 gene of malignant melanoma cells, and it would be useful for the further experiment of the inhibition of the growth of nude mouse transplantable tumor, and it would provide experiment evidence for tumor gene therapy.