MiR-497 Regulates The Mechanism By Which Hif1α Induces The Production Of Inflammatory Factors In Astrocytes Of EAE Mice

Part I Expression and analysis of down-regulated microRNA and up-regulated Hifla both in the brain tissue of mice with EAE and in the astrocytes stimulated by IL-17 in vitroObjective:To study the expression changes of microRNA(miRNA)and transcription factor,namely hypoxia inducible factor 1α(Hif1α)in the brain tissue of EAE mice and astrocytes stimulated by IL-17 in vitro.Method:The model of experimental autoimmune encephalomyelitis(EAE)in mice was established and identified by injection of myelin oligodendrocyte glycoprotein(MOG).At the same time,the primary astrocytes of C57BL/6 mice were also cultured and identified in vitro.Then,the down-regulated expression profiles of miRNAs both in the brain tissue of EAE mice at 14d,and 20d after giving MOG(in vivo)and in the primary astrocytes at 3h and 6h after IL-17 stimulation(in vitro)were analyzed with miRNA microarray,to find out the co-downregulated miRNAs in vivo and in vitro.In addition,the expression level of transcription factor Hif1α both in the brain tissue of mice with EAE on 7d、14d、18d and 20d after MOG immunization and in the primary astrocytes treated with IL-17 at 3h、6h、12h、24h and 48h was also examined using Western blot.Results:(1)On the basis of mice of EAE model establishment and cultured primary astrocytes in vitro,the down-regulated miRNAs were analysed,and the results showed that,in the brain tissue of EAE mice on day14 and day 20,there are 42 miRNAs less than that of control group(0.5 fold),and 62 in the primary astrocytes at 3h and 6h after IL-17 stimulation less than that in the astrocyte untreated with IL-17(0.5 fold).There are 16 co-downregulated miRNAs in vivo and in vitro.(2)The protein expression of transcription factor Hifla was significantly increased both in the brain tissue of EAE mice and in the primary astrocytes stimulated with IL-17.Conclusion:Co-downregulated microRNAs were found in the brain tissue of EAE mice and the primary astrocytes stimulated with IL-17.Meanwhile,the transcription factor Hif1α were shown to co-upregulated both in vivo and in vitro,and the expression phases of Hif1α and microRNA were consistent.PART Ⅱ The effects and mechanisms of miR-497 regulation on the production of inflammatory factors in mouse astrocytes induced by IL-17 stimulation in vitro.Objective:To explore the effects of inducible miRNA regulating Hifla on the production of IL-1βand IL-6 in mouse primary astrocytes triggered by IL-17 stimulation.Methods:According to the results from microRNA array,some miRNAs that may not only combined with Hifla 3’UTR region predicited by bioinformatics software,but also be downregulated in response to IL-17 stimulation were confirmed using Western blot and luciferase reporter assay.Next,the expressions of the selected miRNAs were again verified using Real-time PCR.The production of IL-1β and IL-6 both in the brain tissue of mice with EAE and in the astrocytes upon IL-17 treatment was also detected using Real-time PCR and ELISA.Moreover,the promoter activity of IL-1β and IL-6 in the astrocytes regulated by Hif1α and the potential combination between Hifla protein and DNA sequences in the promoters of IL-1β and IL-6 were measured with luciferase reporter and ChIP assays.Furthermore,when the expression of IL-17RA in the astrocytes was knocked down by transfecting shIL-17RA plasmids in advance,the miRNA or protein level of miR-497,Hif1α and IL-1β as well as IL-6 in the astrocytes stimulated with IL-17 was detected using Real-time PCR or ELISA respectively.And meantime,mRNA and protein level of Hifla,IL-1β and IL-6 were also measured using Real-time PCR and Western blot or ELISA after the astrocytes were tansfected with miR-497 mimics or miR-497 inhibitor,and plasmids of Hif1αoverexpression or siHif1α fragment separately.Results:(1)Downregulated expression of miR-292-5p and miR-497,especially miR-497,could significantly inhibit not only the protein expression of Hifla induced by IL-17,and but also the luciferase activity of pGL3-Promoter/Hifla in HEK293T cells;(2)The promoter activity of IL-1β and IL-6 in the astrocytes could be remarkably increased by Hifla,which could be upregulated by IL-17 stimulation.Hifla protein could directly bind to gene promoters of IL-1β and IL-6;(3)Knockdown of IL-17RA expression could significantly inhibit the downregulation of miR-497 and the upregulation of Hifla,IL-1 and IL-6 expression mediated by IL-17 treatment.In addition,after the expression of miR-497 or Hifla was silenced,induction of Hif1α,IL-1β and IL-6 were markably upregulated,and vice versa;(4)The secretion of IL-1β and IL-6 in the astrocytes after transfection of Hif1αoverexpression plasmids increased obviously,but the production of IL-1β and IL-6 were notably reduced while silencing the Hif1α expression with siHif1α.Conclusion:Transcriptional factor Hif1α,as a target gene of miR-497,could bind to specific elememt on promoters of IL-1β and IL-6 in the primary astrocytes in response to IL-17 stimulation,in which the miR-497 was downregulated whereas expression of Hif1α was upregulated,and consequently facilitated production of IL-1β and IL-6.