Screening Of RNA Interference Seqnences Targeting Against High Mobility Group Box1Protein Gene And Research For It，S Biological Effects

OBJECTIVETo screen the effective small interfering RNA(siRNA) sequenceinhibiting high mobility group box1protein (HMGB1) gene, and observeit’s inhibitory effects for inflammatory response in vitro,which were usedto provide new route for treating animal sepsis.METHODSA total of three siRNA sequences(gene site1550-1570;775-795;1719–1739) which were selected by random selection method were namedsiRNA1、siRNA2、siRNA3. All siRNA sequences were blast in GenBank toconfirm that only HMGB1gene was targeted. All siRNAs were synthesizedby T7transcription system in vitro and transfected to RAW264.7cells. Thecells transfected with the siRNAs were being stimulated by Lipopolysaccharide (LPS) at final concentration of500ng/ml.HMGB1expresssion level was evaluated by reverse transcription Polymerase ChainReaction (RT-PCR) and Enzyme-linked immunoabsorbent Assay (ELISA).The high effective siRNAs was diluted into different concentration andthen transfected to RAW264.7cells. After and before transfected with thesiRNAs,the cells were being stimulated by LPS at final concentration of500ng/ml. We observed that whether HMGB1expresssion and HMGB1protein were inhibited by siRNA in a dose-dependent manner, and TNF-α、IL-1β levels.RESULTSsiRNAs which were synthesized in vitro with T7RNA polymeraseWere detected in3%agarose gel electrophoresis,and the result showed thatRNA fragment length was21bp which is the same as weexpected,implying we sucessed to synthesize siRNAs. HMGB1mRNAexpression and the release of HMGB1protein were inhibited by the threeof HMGB1siRNAs, one of which owned the best inhibitory effect.Toobserve the persistent of HMGB1siRNA inhibitory effect, the cellstransfected with the siRNAs were being stimulated by for24h or48h,andHMGB1expresssion level was evaluated by RT-PCR and ELISA, which ofthe results showed that HMGB1mRNA expression was inhibited andHMGB1release was reduced by HMGB1siRNA, while the defect was not as good as a24-hour time point.The effective siRNA was diluted intodifferent concentration (50nM,100nM,150nM, and200nM) topretreated RAW264.7cells. The siRNA conferred inhibitory activity forHMGB1expression in a dose-dependent manner. Meanwhile, weinvestigated that whether other inflammatory factors were inhibited by thesiRNA.The results showed below: TNF-α、 IL-1β were significantlydecreased. The inhibitory effect to TNF-α was best at this point in time of24h,while the inhibitory effect to IL-1βwas best at this point in time of16h.For assessing the therapeutic efficacy of siRNA, we administratedsiRNA1to RAW264.7cells12h after the stimulation of LPS. the HMGB1expression were inhibited by siRNA1at different concentrations comparedwith treatment with transfection reagent only.CONCLUSIONSA large amount of siRNA was synthesized by T7RNA polymerasetranscription in vitro. This method was simple and rapid,whichsynthesiszed siRNA could effectively reduce the HMGB1and otherinflammatory cytokines so as to mitigate the further development ofinflammation.The siRNA sequence screnned in our experiment is expectedto become effective drug for treating inflammatory diseases, and has acertain potential for development.