Research On The Regulation Of Rab7Gene Silencing On The TLR4/TLR3Signaling Pathways In Macrophages

The family of Toll-like receptors(TLRs) initiates an innate immune response after recognition of pathogen-associated molecular pattems(PAMPs), they are a key position to link innate immune response and adaptive immune response. However, the activation of TLRs have dual character:On one hand, the normal activation of TLRs can induct innate immune response and adaptive immune response, and effectively suppress to invade of pathogenic microorganism; On other hand, the anormal or over-activation of TLRs may cause diseases associate with systemic inflammatory response. Rab7is a novel member of the Rabs famlily, it is specifically localized in some organelle associated with late endosome/lysosome, and regulate the transport from early to late endosome or the fusion early and late endosome, and vesicular traffic. The present study indicate that Rab7maybe also regulate cell transmembrane signal transduction. The article study that the influence of cytokines production in Rab7gene silenced RAW264.7after stimulation with LPS and PolyI:C, and investigate that the regulation of Rab7to TLR4/TLR3signal transduction pathways. Firstly, we designed and synthesised Rab7-siRNA sequences, and were transiently transfected into RAW264.7cell by lipidosome, than detected the interference effect through Real-time PCR and Western blot assay. In addition, we detected the effect of the growth condition of transformed RAW264.7by MTT assay. After Rab7gene was silenced, the cytokines expression were detected by RT-PCR and RTQ-PCR in LPS and PolyI:C stimulated RAW264.7cell at different time points. In the study, we found that transfected Rab7-siRNA3resulted in the lowest Rab7mRNA and protein expression(p<0.05), and Rab7gene silenced significantly promote cell proliferation. After Rab7gene silenced, the mRNA expression of TNF-α、IL-1β、IL-6、 IFN-β、IP-10、NO/iNOS and TLR4obviously increased in LPS stimulated RAW264.7(p<0.01, p<0.05), among TNF-α、IL-1β、IL-6、IFN-β and IP-10achieved to the top at6h; NO and iNOS achived the peak at12h; TLR4achieved the maximum. At the sometime, we also study that after PolyI:C stimulated Rab7gene silenced RAW264.7, the mRNA expresion of IFN-β、IP-10and iNOS increased, but have no significant difference. In the word, Rab7-siRNA was transiently transfection successfully, and we concluded that Rab7gene silenced significantly promote the proliferation of macrophages, and Rab7negative regulate TLR4/TLR3signaling pathways. The research firstly used Rab7-siRNA transient transfection to study the influence of Rab7to TLRs signal transduction pathways, and suggested a novel thread for Rab proteins regulated TLRs signal pathways, it is helpful for finding a new target point of treatment some disease associated with TLRs signal transduction pathways.