Vector-mediated Sirna In Human Ra Synovial Cells, Nf-¦Êb Gene Expression Study

OBJECTIVE:To design and synthesize small interfering RNA(siRNA) targeting human neuclear transcription factor-kappaB(NF-κB),then construct siRNA expression vector(pGenesil-1/siRNA plasmid),and transfect into human rheumatoid arthritis(RA) synovial cells,so as to identify the effective siRNA which could specifically block the NF-κB production.Which can provide theoretical basis for RA gene therapy by NF-κB small RNA interfering.METHODS:RA synovial cells were isolated and digested from RA synovectomy tissue,then cultured,passed for use.Four specific siRNA lines(1#～4#siRNA) and a scrambled line as control were designed and synthesized according to GeneBanK NF-κB mRNA full-length open reading frames(ORFs)(L19067),and verified by the thumbs-up rules,analyzed by basic local alignment search tool(BLAST) to ensure that they have no significnat sequence homology with other genes.Then the four siRNA lines and a scrambled line were reconstructed into expression vector pGenesil-1.To generate NF-κB targeting siRNA expression vector pGenesil-1/NF-κB.The synovial cells were transfected with recombinant siRNA targeting NF-κB expression vector pGenesil-1 /NF-κB by liposome-mediated gene transfer method.The synovial cells were collected respectively 12h,24h,36h,72h,5d,7d after transfection.The total RNA were extracted for real-time fluorescent quantitive polymerase chain reaction(FQ-PCR) measurement (specific primer from ORFs of NF-κB mRNA were designed and synthesized according to GenebankL19067).RESULTS.After transfection 12h,24h,36h,72h,5d,7d,the NF-κB mRNAlevels of the synovial cells transfected with 3#pGenesil-1/NF-κB siRNA were significantly lower than blank control,scrambled siRNA group,1# pGenesil-1/NF-κB siRNA,2# pGenesil-1/NF-κB siRNA,4#pGenesil-1/NF-κB siRNA group.CONCLUSION:3#pGenesil-1/NF-κB siRNA could effectively interfering human RA synovial cells NF-κB mRNA expression.It will provide experimental and theoretical support for RA gene therapy by small RNA interfering NF-κB.