Knock-down of NF-κB p65 Expression by Recombinant Adenovirus-delivered siRNA in Rat Hepatocytes

Objective To construct recombinant adenovirus containing small interfering RNA(siRNA) targeting rat NF-κB p65 mRNA(Ad-NF-κB p65-siRNA),and to evaluate its potential of suppressing NF-κB p65 expression in rat hepatocytes in vitro.Methods(1) The specific siRNA sequence targeting rat NF-κB p65 mRNA was selected,and the homologous double-strand DNA was designed and synthesized,then the DNA was inserted into plasmid pShuttle-H1,and psiRNA-NF-κB p65 was obtained.The psiRNA-NF-κB p65 was co-transfected into the E.coli strain BJ5183 with the bone plasmid pAdEasy-1,and then Ad-NF-κB p65-siRNA was generated by homologous recombination.After being identified by restriction digest and gel electrophoresis technique,the recombinant Ad-NF-κB p65-siRNA was packaged and amplified in cells 293 A.(2) Ad-NF-κB p65-siRNA was transfected into rat hepatocytes BRL cells.The content of NF-κB p65 mRNA and protein in the cells was tested by reverse transcription-PCR and Western blot respectively 48 hours later.Results The recombinant Ad-NF-κB p65-siRNA was successfully constructed.After being infected by the Ad-NF-κB p65-siRNA,the NF-κB p65 mRNA and protein content in BRL cells was decreased by 81% and 71% compared with that in the mock infected control group.Conclusion Constructed Ad-siRNA-NF-κB p65 can efficiently decrease the NF-κB p65 expression in BRL cells in vitro.