Construction of MEKK2 siRNA Recombinant Adenovirus Vector and Its Effect on the Inhibition of MEKK2 Expression

Abstract: Three different siRNA template DNA sequences of MEKK2 gene were designed by Genscript siRNA design software. The corresponding DNA fragments were synthesized in vitro， annealed and then cloned into the pRNAT-H1.1/Adeno shuttle vector. The recombinant shuttle vectors were confirmed by DNA sequencing， then transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdEasy-1 to obtain adenovirus plasmid through homologous recombination. The adenovirus plasmid was transfected into 293A cells to form adenovirus particle. Followed， the adenovirus particles were transduced into AGS cells. Western blot was carried out to analyze the suppression effect of MEKK2 adenovirus expression vectors in AGS cells and to screen the best vector that had the highest effect on inhibition of MEKK2 expression. DNA sequencing confirmed that the MEKK2 siRNA adenovirus expression vector were successfully constructed and the results of Western blot showed that one of the three MEKK2 siRNA adenovirus vectors (pAdeno-MEKK2 siRNA2， targeting MEKK2 cDNA sequences of 992-1 010) could effectively silence MEKK2 expression in AGS cells. No obvious inhabition effect was found in pAdeno- MEKK2 siRNA1 (targeting MEKK2 cDNA sequences of 1 456-1 474) and pAdeno-MEKK2 siRNA3 (targeting MEKK2 cDNA sequences of 1 351-1 369). In conclusion， the recombinant pAd-MEKK2-siRNA expression vector targeted on MEKK2 was successfully constructed， which can effectively suppress MEKK2 expression in AGS cells. The apoptosis induced by H2O2 was significantly lower， whereas cell survival number was significantly higher in MEKK2 knocked down AGS cells than those in transduced siRNA negative control cells or wild type cells (P<0.05). This study will provide a foundation for further investigation on the function of MEKK2 gene in gastric adenocarcinomas cells.

CSTR: 32200.14.cjcb.2012.03.0008