The Preliminary Study Of Mechanisms For SIGIRR Regulating NF-κB Activation And The Development Of EMT As Well As Feedback Control Of NF-κB Toward SIGIRR Expression In Human Renal Tubular Epithelial Cells

Background&Objective: Lupus nephritis(LN),whose etiology and pathogenesis have not been clearly elucidated so far, is one of the most common complications of systemic lupus erythematosus(SLE), additionally,the severity of LN has a significant impact on mortality and life expectancy of patients with SLE. More and more studies have reported that the Toll-like receptor/interleukin-1 receptor(TLR/IL-1R) signaling pathways involved in the pathogenesis of SLE. Many evidences have shown that single immunoglobulin interleukin 1 receptor related protein(SIGIRR), also known as the Toll/interleukin-1 receptor8(TIR8), a member of the TLR/IL-1R family, present a negative signal for TLR/IL-1R pathway,which may be a potential therapy target of LN, but the regulating mechanism of SIGIRR expression have not been clear. Nuclear transcription factor NF-κB(NF-κB),a kind ofnuclear transcriptional regulatory elements,has the ability ofregulating transcription and translation of a variety of genes after activated. TLR/IL-1R-NF-κB pathway overactivation plays an important role in the regulation of inflammatory mediators and cytokines in immunity-mediated renal damage including LN.The actional mechanism of many clinical drugs is to interfere the activationof NF-κBtoameliorate the development of immunity-mediated disease. Therefore,we will explore in the human renal tubular epithelial cells that weather SIGIRR can regulate NF-κB(p65) excessive activation and activated NF-κB(p65), inturn, can regulate the expression of SIGIRR to maintain self immune tolerance. Additionallynot only does renal interstitial fibrosis(RIF) play an important role in theprogression of terminal renal failure in LN and other chronickidneydisease,but also renal tubularepithelial-mesenchymal transdifferentiation(EMT), also known as renal tubular epithelial cells into myofibroblasts, is the key event occurred for RIF.Work by many groupssuggested thatexcessive activation of interleukin-lβ(IL-1β) had the capacity to induce tubular epithelial cells into myofibroblasts through TLR/IL-1Rpathway in patients with LN,which play an important role in the RIF, whereas SIGIRR has a negative regulatory role in TLR/IL-1R pathway, so we will questionedwhether SIGIRR can block IL-1β-induced TLR/IL-1R-NF-κB signaling pathway activation to amilerate the development oftransdifferentiation of human renal tubular epithelial cells.Methods: Part I: Designed sh RNA sequences,a target for the SIGIRR gene, connected with the GV248-GFP-Puro to produce recombinant lentiviral vector(GV248-GFP-Puro-sh SIGIRR). The corrected recombinants, identified by sequenced, were transfected into 293 T cells with packaging plasmids(p MDL, p Rev and p VSVG) for virus packaging. HKC cells were then infected by packaged viruses. Quantitative real-time PCR(q RT-PCR)and western blot were used to detect the interference efficiency of SIGIRR in HKC cells. The phosphorylation level of NF-κB(p65) as well as m RNA expression levels of monocyte chemotactic protein 1(MCP-1) and regulated upon activation normal T cell expressed and secreted factor(RANTES) in both SIGIRR stable knockdown HKC cells(HKC/sh SIGIRR) and the control cellsafter stimulated with IL-1β were detected by western blot or q RT-PCR. Part II: The wild-type HKC as a model to build p65 stable knockdown HKC cells(HKC/shp65), RT-PCR, western blot and immunofluorescence were used to detect the expression of SIGIRR in both HKC/shp65 and HKC cells. Online prediction show that NF-κB(p65) can bind to SIGIRR promoter region, which was checked by CHIP in wide HKC cells. Humanwhole genome DNA were extracted from 293 T cells, and obtained SIGIRR promoter sequences by PCR method then connected to p GL3-Basic luciferase reporter gene vectors. Restriction enzyme digestion and sequencing were used to teset successfully constructed, then mutated the DNA of NF-κB(p65) bind to SIGIRR promoter to construct site-directed mutagenesis, and then the luciferase reporter gene assay indirectly further validation NF-κB(p65) can be combined with the expression and regulation of SIGIRR. Part III: The people SIGIRR c DNA fragments were ligated to the retroviral vector p LNCX2-G418, HKC cells were then infected by packaged viruses. Quantitative real-time PCR(q RT-PCR)and western blot were used to detect the expression of SIGIRR in HKC cells. SIGIRR stable overexpression HKC cells(HKC/SIGIRR) and the control cells were induced by IL-1β, cell morphology was observed under an inverted microscope, EMT associated molecular markers(E-cadherin and Vimentin) were detected by western blot and immunofluorescence, cell migration were detected by scratch test and Transwell assay, the phosphorylated NF-κB(p65) expression was detected by western blot after extracting nucleoprotein, and the m RNA expression levels of transforming growth factor-β(TGF-β) m RNA expression was analyzed by q RT-PCR.Results: Part I: The results showed that the recombinant lentiviral vector GV248-GFP-Puro-sh SIGIRR was successfully constructed. q RT-PCR and Western blot confirmed that SIGIRR was successfully knockdown in HKC cells. Moreover, the results also show that, the phosphorylated NF-κB(p65) and the m RNA levels of MCP-1 and RANTES were significantly increased in HKC/sh SIGIRR cells compared with the control cellsafterstimulated with IL-1β. Part II: The results showed that SIGIRR protein expression was down-regulated when p65 was knockdown in HKC cells. Online prediction showed that p65 could bind to SIGIRR promoter. Checked by CHIP. p GL3-SIGIRR-Luc and mutation carriers were successfully constructed, and the luciferase assay proved a further evidencethat p65 could bind to the promoter region ofSIGIRR and in turn can regulate its expression. Part III: The results showed that the recombinant retroviral vector p LNCX2-G418-SIGIRR was successfully constructed. q RT-PCR and western blot confirmed that SIGIRR was overexpression in HKC cells. Moreover, the results also showed that compared with the HKC/SIGIRR cells, the control group cells were spindle shape change, epithelial cell marker E-cadherin expression decreased, mesenchymal cells marker Vimentin expression increased, and cell migration also enhanced by stimulated with IL-1β. It is found that the effect is mediated by SIGIRR to affect activation of TLR/IL-1R-NF-κB signalinginduced by IL-1β to reduce the TGF-β produce implementation.Conclusions: The first part results suggested that SIGIRR acted as a "brake" of inflammatory reaction in Toll-like Receptor/IL-1 Receptor(TLR/IL-1R) pathway in HKC cells, and in the second part found that activation NF-κB(p65),TLR/IL-1R signaling pathway downstream of the target molecules, may in turn regulate the expression of SIGIRR to maintain the immune balance. In the third part the results suggested that SIGIRR could reduce IL-1β induced EMT through the regulation of TLR/IL-1R-NF-κB signaling pathway.