The Effect And Mechanism Of Histone Demethylase (KDM6A) On Migration And Invasion In Glioblastoma

Glioma is the most common primary intracranial tumor,which is divided into four grades according to the degree of malignancy:I-IV.glioblastoma（GBM）is the most malignant grade IV glioma.GBM patients have a poor prognosis and low survival rate.The standard treatment for postoperative GBM patients is radiotherapy combined with chemotherapy;however,the average survival rate for GBM patients is still less than 20months.Therefore,it is important to explore the pathogenesis of GBM and find new therapeutic targets.KDM6A（also called UTX）,a histone methylation enzyme gene.It contains a Jmj C domain with histone H3 lysine 27（H3K27）demethylase activity.KDM6A plays an important role in bladder cancer,breast cancer and other diseases.High aggressiveness is one of the important characteristics of GBM,and the hypoxic environment is closely related to the aggressiveness of GBM.Hypoxia stabilizes Hif1αexpression,activates Snai2/CDH1 axis,promotes migration and invasion,and increases tumor drug resistance.Although KDM6A has been found to inhibit cell migration and invasion in a variety of tumors and inhibit cancer development,little research has been conducted in GBM.This thesis focuses on exploring the role of KDM6A in the development of GBM,analyzing the impact of KDM6A expression on the prognosis of GBM patients through the prognostic database,exploring the impact of KDM6A on the proliferation and tumorigenic ability of GBM cells by various experimental methods,and exploring the molecular mechanism of KDM6A regulation of GBM migration and invasion.Finally,it is explained that KDM6A affects GBM migration and invasion through Hif1α-Snai2.It enriches the research of KDM6A in GBM and provides new ideas for GBM treatment.So far,this paper has achieved the following results:1.Overexpression of KDM6A inhibits glioblastoma growth and proliferationTo explore the role of KDM6A in GBM,we analyzed the prognosis of KDM6A in GBM patients.We found that low expression of KDM6A predicted poor prognosis of GBM patients,which implied that KDM6A could be used as a potential prognostic indicator.In order to explore the effect of KDM6A on the proliferation of glioblastoma cells.First,we first examined the interference efficiency of the sh KDM6A fragment and the effect of overexpressing KDM6A（OE-KDM6A）.Subsequently,MTT assay and Edu assay were used to detect the effect of overexpression of KDM6A on GBM proliferation,and it was found that KDM6A overexpression could significantly inhibit GBM proliferation.Plate cloning assay,soft agar assay and in situ tumor formation assay were used to detect the effect of overexpression of KDM6A on GBM tumor formation in vivo and in vitro,and it was found that overexpression of KDM6A significantly inhibited the tumor formation ability in vivo and in vitro.We subsequently examined the effect of KDM6A on GBM cell cycle progression and found that overexpression of KDM6A resulted in cell cycle arrest in G1 phase and found that the expression of cyclin in G1phase was down-regulated.GSEA enriched signaling pathways related to KDM6A expression and found that low KDM6A expression was associated with GBM cyclin CCND1 expression.Over-activation of AKT signaling pathway leads to abnormal tumor proliferation.In order to explore the regulation of AKT signaling pathway by KDM6A.GSEA enrichment analysis examined the enrichment of KDM6A in AKT signaling pathway,and found that KDM6A low expression was positively enriched with AKT signaling pathway.By examining AKT signaling pathway proteins,it was found that KDM6A overexpression inhibited AKT signaling pathway,while KDM6A interference promoted the activation of AKT signaling pathway.Quantitative PCR assay of AKT upstream inhibitor showed that overexpression of KDM6A promoted the activation of AKT upstream inhibitor.In conclusion,high expression of KDM6A inhibited glioblastoma proliferation,caused G1 phase arrest and inhibited AKT signaling pathway.2.Overexpression of KDM6A inhibits cell migration and invasion in glioblastomaIn order to explore the effect of KDM6A on the migration and invasion of glioblastoma,Transwell assay was used to detect the effect of KDM6A overexpression on the migration and invasion of glioblastoma.The results showed that KDM6A significantly inhibited the migration and invasion ability of glioblastoma.Next,the effect of KDM6A on glioblastoma migration was detected by scratch assay,and the results showed that KDM6A overexpression significantly inhibited the wound healing rate of glioblastoma.Western blot was used to detect the effect of KDM6A on EMT process-related proteins in glioblastoma,and the results showed that KDM6A significantly inhibited EMT process-related proteins in glioblastoma.Then,by interfering with KDM6A,we examined the changes in EMT process proteins and transcript levels,and the results showed that KDM6A interference could activate the expression of these genes.To further explore the effect of KDM6A on the EMT process of glioblastoma,glioblastoma cell lines were treated with GSKJ-4（KDM6A inhibitor）,and Transwell and scratch assays were used to detect the effect of GSKJ-4 on the EMT process of glioblastoma.GSKJ-4 can enhance the migration and invasion ability of glioblastoma,which is the same as the previous conclusion,indicating that KDM6A inhibition leads to the activation of EMT process in glioblastoma.Then,the effect of GSKJ-4 on EMT process of glioblastoma was detected by Western blot assay.The results showed that GSKJ-4 activated Snai2 and inhibited CDH1 expression in EMT process.The above results indicate that overexpression of KDM6A can inhibit the migration and invasion of glioblastoma,on the contrary,KDM6A interference and GSKJ-4 can promote the migration and invasion of glioblastoma,indicating the important role of KDM6A in regulating the migration and invasion of glioblastoma.3.KDM6A regulates GBM migration and invasion through the Hif1α-Snai2 axisAnoxic microenvironment is a common phenomenon in tumors.The occurrence of anoxic environment will increase the invasion of tumors and promote the proliferation and deterioration of tumors.In order to explore the influence of KDM6A on anoxic environment,Co Cl₂ treated GBM cells to simulate anoxic environment.Western blot and RT-q PCR were used to detect the influence of anoxic environment on KDM6A,and it was found that the expression of KDM6A was inhibited in anoxic environment.The effect of KDM6A on Hif1αwas detected by Western blotting,and it was found that KDM6A inhibited the expression of Hif1α,and RT-q PCR showed that KDM6A inhibited the transcription of Hif1αand its downstream genes.To further explore the regulation of KDM6A on Hif1α,CHIP experiment found that KDM6A transcription binding Hif1αpromoter,double luciferase reporter gene assay to detect the effect of KDM6A on Hif1αpromoter activity,it was found that KDM6A inhibited Hif1αpromoter activity.The above results showed that the expression of KDM6A decreased under the influence of hypoxia environment,while the expression of Hif1αincreased.Moreover,KDM6A can transcriptionally inhibit Hif1αand bind the promoter of Hif1α.Previously,we explored that KDM6A had a regulatory effect on Hif1α,and found that Snai2 was a downstream gene of Hif1αbased on literature and database.The effect of Co Cl₂-induced hypoxia on Snai2 and CDH1 was examined by immunoblotting,and it was found that hypoxia activates SNAi2-CDh1 signaling.In order to explore the effect of KDM6A on Snai2,the effect of KDM6A on Snai2 was detected by immunoblotting and RT-q PCR,and it was found that KDM6A inhibited the expression of Snai2.Subsequently,chip-seq data were used to analyze the binding of KDM6A to the promoter region of Snai2,and CHIP experiments found the site of KDM6A transcription binding to Snai2.Dual luciferase reporter assay was used to examine the effect of KDM6A on Snai2 promoter activity and found that KDM6A transcription inhibited Snai2.To further investigate whether KDM6A inhibits GBM migration and invasion through Snai2,Snai2 was interfered in GBM cell lines interfering with KDM6A,and it was found that the enhanced migration and invasion ability caused by interfering with KDM6A was re-inhibited by interfering with Snai2.Taken together,these conclusions suggest that KDM6A inhibits GBM migration and invasion by inhibiting Snai2 transcription.In summary,we found that KDM6A inhibited GBM proliferation by inhibiting GBM cell cycle progression and AKT signaling pathway.KDM6A is affected by the hypoxia environment simulated by Co Cl₂,and KDM6A can bind the Hif1αpromoter to inhibit the transcription of Hif1αand inhibit the EMT pathway induced by hypoxia.KDM6A interacts with Snai2 and inhibits GBM migration and invasion by inhibiting Snai2transcription.