Construction And Identification Of MicroRNA Eukaryotic Expression Vectors Targeting Vsacular Endothelial Growth Factor

ObjectiveVascular endothelial growth factor(VEGF)has been implicated as a critical molecular signalin tumour development,by promoting angiogenesis and possibly by exerting autocrine functionsin tumour cells.Some studies showed that VEGF was over expressed in hepatocarcinoma. Howto inhibit effectively the expression of VEGF is the emphasis of tmuor treatment.MicroRNA(miRNA) is a small non-coding RNA that cantains 21 to 23 nucleotides and cannegatively regulate gene expression in animals,by binding,with imperfect base pairing,to targetsites in messenger RNAs(usually in 3'untranslated regions)thereby either reducing translationalefficiency or determining transcript degradation. Recent studies found that some miRNAs mightfunction both as oncogenes and tumor suppreesors; its role in the tumorigenesis maycomplenment and enrich the mechanisms of tumorigenesis. Recently a large number of studiesshow that amiRNA,which targeted specific gene,can be introduced into cells through microRNAexpression framework,and can inhibit the expression of target genes. Therefore,this experimentalstudy constructed miRNA expression plasmids which targeted VEGF gene to transientlytransfected HepG2 cells.We observed whether miRNA-mediated RNA interference could blockVEGF gene expression by measuring the inhibition effect of the target gene and VEGF proteindecrease level,so that we could understand whether miRNA mediated RNAi targeting VEGFgene can be used in hepatocarcinoma gene therapy.Methods(1) Designed and synthetized sequences: Searched and found the mRNA sequence of VEGFgene (Genebank ID, NM₀01025366) from GenBank. Used the online application software,offered by Invitrogen company, to design four VEGF pre-microRNAs oligonucleotidemicroarray and got there complementary sequences, which were excluded the nonspecific homology by NCBI Blast. And also designed a negative control sequence (Neg), thisnegative control not aimed at any mRNA sequences of the human body.(2) Constructed the eukaryotic vector: Inserted these sequence synthesis into the eukaryoticexpression vector, pcDNA6.2-GW/EmGFP-miR, respectively and transfected them intoDH5 alpha feeling state e. coli. Extracted plasmid and purified, sequenced the positivecloning that had preliminary identification. Recombinant named VEGF-miRNA-1,VEGF-miRNA-2, VEGF-miRNA-3, VEGF-miRNA-4 and VEGF-miRNA-Neg respectively.(3) Transfected into hepatocarcinoma cells: Used liposome carrying method to transfect fourrecombinant plasmids into HepG2 hepatocarcinoma cells respectively and the blank contrastonly transfect the Lipofectamine 2000. After 24 hours, observed the EmGFP expression inHepG2 cells through inverted fluorescence microscope and evaluated there transfectionefficiency.(4) Validated transfection efficiency: Quantitative polymerase chain reaction (qPCR) detectedthe difference expression level of VEGF mRNA in hepatocarcinoma cells, testified to thespecificity inhibitional effect of miRNA– VEGF to target genes, and specified that whichrecombinant plasmid had the strongest inhibitional effect and its miRNA sequence.Results(1) Successfully constructed four MicroRNA eukaryotic expression vectors of VEGFmRNAsequences;(2) VEGF-miRNA-1,VEGF-miRNA-2,VEGF-miRNA-3,VEGF-miRNA-4interferingveetors allinhibit VEGFmRNA expression and the inhibition rate of VEGF-miRNA-3 is 87%. Theinhibition rate of VEGF protein of VEGF-miRNA-3 is 87% detected by western blotting.ConclusionSuccessfully constructed MicroRNA eukaryotic expression vector targeting VEGF, and theexpression vector could significantly inhibit the expression of VEGF in HepG-2 cells. Theresearch provided the theoretical and experimental basis for treating hepatocarcinoma with RNAimethods.