Construction Of DJ-1-shRNA Eukaryotic Expression Vectors And Its Effects On DJ-1in The Transfected SGC7901Gastric Cancer Cells

Objective：To construct a siRNA expression plasmid transcripted by RNA interference,using gene DJ-1as a clone target. To identify the recombinant plasmid nucleic acidsequence by sequencing on the standard sequencer, the recombinant vectors weretransfected into SGC7901Gastric cancer cells，and to evaluate the plasmid impact tothe DJ-1gene expression in SGC7901. These results lay a foundation for the furtherresearch of DJ-1is involved in gastric cancer cell migration and invasion, laid thebasis of the pre-trial to establish the animal model study the molecular mechanism ofperitoneal metastasis of gastric cancer.Methods：The analytical sequence of human DJ-1gene were retrieved from Genbank.According to the guidelines for http://www.ambion.com/techlib/misc/siRNA finder.html, three targeting DJ-1genes sequence of candidate RNA interference weredesigned,and construct recombinant plasmid by ShRNA eukaryotic expression vectorpGPU6/GFP/Neo recombinant plasmid; design and build the negative control RNAinterference recombinant vector used to identify the specificity of the pGPU6/GFP/Neo plasmid-mediated RNA interference system.The synthesized sequences werecloned into a vector pGPU6/GFP/Neo and identified by sequencing analysis ofnucleic acid. The recombinant plasmid was transformed into E.coli Top10forcloning.Subsequently,recombinant plasmid was transformed into the competent cellsSGC7901with lipofectamine2000, The expression of the DJ-1gene mRNA andprotein on the transfected cells was measuredby RT-PCR and western blot.Results：The strain E.coli Top10transformed by recombinant plasmid could clony growby ampicillin-resistant screen.The DNA sequence analysis showed that the threerecombinant vectors and the negative control RNA interference recombinant vectorwere constructed exactly, termed as pGPU6/GFP/Neo-shDJ-1-A， pGPU6/GFP/ Neo-shDJ-1-B， pGPU6/GFP/Neo-shDJ-1-C and pGPU6/GFP/Neo-shNC. Theresultant RT-PCR product was detected after transient transfection of target genespGPU6/GFP/Neo-shDJ-1-C transfection group DJ-1expression strongest inhibitoryeffect of up to78%, two groups of the negative control recombinant vector wastransfected DJ-1gene expression was not change significantly(P<0.01). The result ofwestern blot showed that after transient transfection of target genes pGPU6/GFP/Neo-shDJ-1-C transfection group DJ-1expression strongest inhibitory effect of up to75%,two groups of negative control recombinant vector was transfected DJ-1geneexpression was not change significantly (P<0.01).Conclusion：construct the recombinant vector DJ-1-shRNA，the pGPU6/GFP/Neo-shDJ-1-Ccould significantly decrease the expression of DJ-1gene mRNA and protein level inSGC7901Gastric cancer cells.