Mechanism For RAI14 Regulating Inflammatory Effects Of Hypoxia-related Astrocytes By MTOR Signaling

Objective:Retinoic acid-induced 14 is a developmentally regulated gene induced by retinoic acid and is closely associated with NIK/NF-?B signaling.In the present study,we examined the effect of RAI14 on mTOR-mediated glial inflammation in response to inflammatory factors and chemical ischemia.Methods:(1)U87cells were incubated for 6,12,24,or 48 h in the presence of PBS(control),LPS(50,100,and 200 ng/mL),or TNF-?(5,10,20,and 50 ng/mL)for the subsequent quantification of RAI14.RT-qPCR and Western blotting were used to measure the level of RAI14 expression across different the times points in response to LPS or TNF-a stimulation.(2)U87 Cells were transfected with siRNA against RAI14(siR-RAI14)or GAPDH(siR-GAPDH)or a Lipo6000TM Transfection reagent(control)as indicated.RT-qPCR was used to measure the level of IL-1? and IL-6 mRNA expression.(3)Immunofluorescence microscopy was used to investigate the potential effect of an RAI14 knockdown on NF-?B p65 activation in U87 cells.Dual luciferase assay was used to measure NF-?B activity.The structural association between endogenous RAI14 and NF-?B p65 was detected by Co-IP.(4)Before treatment with everolimus(20 nM),U87 cells were exposed to LPS(200 ng/mL)for 6 h or TNF-?(20 ng/mL)for 48 h and the level of RAI14 protein expression was analyzed.Effect of everolimus(20 nM)exposure on mRNA levels for RAI14 and inflammatory cytokine in U87 cells following LPS stimulation can be analysed by RT-qPCR.Immunofluorescence microscopy was used to analyze protein levels of p-mTOR and p-eIF4EBP from U87 cells cultured with everolimus(20 nM)for 24 h.(5)U87 cells were pre-treated with LPS(200 ng/mL)for 6 h before plasmid transient transfection for 6 h.Levels of the pro-inflammatory cytokines,IL-1(3,IL-6,and TNF-a,were quantified by RT-qPCR.After transient transfection,U87 cells were exposed to LPS(200 ng/mL)for 6 h or TNF-??(20 ng/mL)for 48 h prior to everolimus(20 nM)treatment for 24 h.The level of p-IKK?/?(Serl76/180)and NF-?B p65 protein expression was analyzed.(6)Following the incubation with 50,100,300,or 500 M CoCl2 for 24 h,U87 cells were lysed to monitor the level ofRAI14 protein expression.Effect of CoCl2 on IL-6 and TNF-a genes expression in U87 cells was analyzed by RT-qPCR.(7)U87 cells were exposed to TNF-?(20 ng/mL)for 48 h prior to artemisinin(20 uM)treatment for 12 h.The level of RAI14 protein expression was analyzed.U87 cells were pre-treated with TNF-?(20 ng/mL)for 48 h before RAI14 knockdown.Levels of the pro-inflammatory cytokines such as IL-1? and IL-6 were quantified by RT-qPCR.Results:(1)Each pro-inflammatory agonist spatially and temporally resulted in the elevation of RAI14 at the mRNA level.The more potent effects were evoked by LPS compared with TNF-a.(2)After an RAI14 knockdown,a significant decrease in RAI14 protein and mRNA expression was observed.A predefined dose of 20 ng/mL TNF-a was shown to significantly promote inflammation in comparison to the control groups.However,the depletion of RAI14 in U87 cell lines resulted in an attenuation of TNF-a-induced inflammation.(3)The inhibition of RAI14 substantially blocked Firefly luciferase activity and siR-RAI14 treatment decreased NF-?B p65 in the nuclei of the stimulated cells.Although RAI14 partially co-localized with NF-?B p65 in the nucleus and the cytoplasm,no structural association between endogenous RAI14 and NF-?B p65 detected by Co-IP.(4)Everolimus reduced the expression of total RAI14 protein,but no significant differences observed for the level of RAI14 mRNA.The mTOR inhibitor significantly inhibited levels of IL-1(3 and IL-6 mRNA and protein levels of p-mTOR but increased protein levels of p-eIF4EBPl.(5)Following siR-RAI14 transfection,IL-1?,IL-6 and TNF-a were observed to be consistently expressed at a lower level than the corresponding controls.Importantly,transfection with siR-RAI14 further strengthened the effects of the mTOR inhibitor on the negative regulation of NF-?B p65 and substantially enhanced the downregulation of everolimus-mediated p-IKK?/?.(6)50 uM CoCl2 resulted in a reduced expression of the total RAI14 protein,while 100 uM CoCl2 resulted in the barely detectable quantification of RAI14 protein levels in U87 cells.Most importantly,this influence reversed by treatment with 300 or 500 uM CoCl2.Consistent with this observation,the impact of CoCl2 on IL-6 and TNF-a gene expression exhibited a similar effect in U87 cells during the same period.Similarly,everolimus was observed to be an effective negative regulator of NF-?B p65 which was further strengthened following transfection with siR-RAI14.(7)Artemisinin reduced the expression of RAI14 protein and inhibited the level of IL-6 and IL-1? mRNA.Importantly,transfection with siR-RAI 14 further strengthened this downregulation effect.Conclusion:RAI14 is the active intermediate responsible for increasing the effect of mTOR on NF-?B activation.(1)The RAI14 appears to be regulated by pro-inflammatory agonists,particularly LPS.(2)RAI14 positively regulates the expression of pro-inflammatory cytokines in response to LPS and TNF.(3)RAI14 is involved in the regulation of the positive response for pro-inflammatory cytokines via NF-?B signaling.(4)The mTOR/eIF4EBP1 signaling is involved in the RAI14 transcription-translation reaction in astrocytes.(5)The mTOR activity may be a strong promoter of the RAI 14-mediated positive regulation of the NF-?B signaling pathway.These results provide insight into the role of RAI 14 on mTOR-mediated NF-?B inflammatory signaling under inflammatory stress and chemical hypoxia.