The Role And Mechanism Of Demethylase ALKBH5 In Regulating Bladder Tumorigenesis

BackgroundBladder cancer is the most common urological tumor,with more than 550,000 new bladder cases and 200,000 deaths worldwide every year,ranking as the 10th most common malignancy worldwide.Despite the availability of novel early diagnostic and progressively advancing treatment methods,bladder cancer remains to be the main cause of tumor-associated mortality.This is due to its high recurrence rates and invasiveness.Therefore,further research into the molecular mechanism underlying the pathogenesis of bladder cancer is urgently in demand.Bladder cancer progression is a complex process.In the tumor malignant progression,it is often found that various mechanisms of human body are disturbed.N6-methyladenosine(m6A)as the most common internal RNA modification,affects all aspects of RNA metabolism such RNA stability,splicing,translation,and transport.Numerous studies have reported that the dysregulation of m6A is widely involved in tumorigenicity and tumor progression.Human AlkB homolog H5(ALKBH5)has been reported to be involved in various cancers,where it exerts demethylase activity.Thus,it is necessary to further explore the role of ALKBH5 in bladder cancer and the regulatory mechanism of ALKBH5-mediated demethylation in bladder cancer.Numerous studies have demonstrated that microRNAs(miRNAs)can regulate tumorigenesis and pathological processes by either inducing mRNA degradation or inhibiting mRNA translation.As a tumor suppressor,miR-200b plays an important role in cell migration,invasion and proliferation.In addition,previous studies have focused on elucidating the mechanism by which demethylases regulate tumor progression by regulating the binding of primary miRNA(primiRNA)to DiGeorge syndrome critical region 8(DGCR8).However,whether ALKBH5 promotes bladder cancer cell proliferation,migration and invasion by regulating miRNA synthesis remains unclear.Zinc Finger E-Box Binding Homeobox 1(ZEB1)is an oncogene that can promote cancer cell proliferation,migration and invasion.ZEB1 functions as an activator of Epithelial-Mesenchymal Transition(EMT),which in turn promotes cancer cell dissociation from the primary carcinoma to facilitate cancer cell migration and invasion to distant sites.In addition,m6A modification has been shown to regulate cancer progression through EMT.Therefore,it is worth further investigating whether ALKBH5-mediated demethylation modification can affect the malignant progression of bladder cancer by regulating EMT.Objective1.To clarify the expression of ALKBH5 in bladder cancer and the effect of ALKBH5 on the migration,invasion and proliferation of bladder cancer cells.2.To explore the mechanism by which demethylase ALKBH5 regulated the synthesis of miR-200b.3.To verify that ALKBH5 regulated the ZEB1 expression by mediating miRNA maturation.Methods1.ALKBH5 expression in bladder cancer and adjacent tissues were detected using Western blotting and IHC.The relationship between ALKBH5 expression and overall survival was analyzed using the Gene Expression Profiling Interactive Analysis(GEPIA)database.2.The association between ALKBH5 expression and EMT-related markers were analyzed using Western blot.We performed Transwell and Wound healing assays to explore the effect of ALKBH5 on the bladder cancer cells migratory and invasive abilities.3.Colony formation assays,CCK-8 and EdU assays demonstrated that the effect of ALKBH5 on bladder cancer cells proliferation and tumorigenesis.4.According to LinkedOmics,we predicted and screened miRNAs in bladder cancer that were negatively correlated with the expression level of ALKBH5.The relationship between the expression of miR-200b,primiR-200b and premiR-200b and the expression level of ALKBH5 were detected using RT-qPCR.RIP and m6A RIP further demonstrated that ALKBH5 regulates the maturation of miR-200b in an m6A-dependent manner.5.Transwell assays demonstrated that the effects of ALKBH5 overexpression and knockdown on the migratory and invasive abilities were reversed by miR-200b mimics and inhibitors,respectively.CCK-8 and EdU assays confirmed that transfection with the miR-200b partially reversed the potentiating effects of ALKBH5 on cancer cells proliferation.6.We used the TargetScan and miRDB databases to predict the target genes of miR-200b.The dual-luciferase reporter assay further confirmed that ZEB1 was the target gene of miR-200b.Western blot demonstrated the effect of miR-200b on ZEB1 expression level and miR-200b partially reversed the effect of ALKBH5 on ZEB1 expression level.7.ZEB1 expression in bladder cancer and adjacent tissues were detected using Western blot.According to the GEPIA database,we analyzed the association between ZEB1 expression and DFS and the association between ZEB1 expression and pathological stage.In the mouse xenograft model tissues,the relationship between ZEB1 and ALKBH5 expression level was detected using Western blot and IHC.Result1.ALKBH5 expression is upregulated in bladder cancer tissues and associated with poor prognosis.2.ALKBH5 regulates EMT progression,ALKBH5 overexpression promotes bladder cancer cells migration and invasion.ALKBH5 knockdown inhibits bladder cancer cells migration and invasion.3.ALKBH5 overexpression promotes bladder cancer cells proliferation.ALKBH5 knockdown inhibits bladder cancer cells proliferation.4.ALKBH5 regulates miR-200b synthesis in an m6A-dependent manner.5.miR-200b partially rescues the ALKBH5-induced proliferative and invasive capabilities of bladder cancer cells.6.ALKBH5 targets ZEB1 through the ALKBH5/miR-200b/ZEB1 pathways in bladder cancer.7.ZEB1 is highly expressed in bladder cancer and positively correlated with ALKBH5.ConclusionThe present study clarified the molecular mechanism of ALKBH5 regulating bladder cancer progression through ALKBH5/miR-200b/ZEB1 axis.ALKBH5 overexpression suppressed miR-200b maturation by interacting with DGCR8,which subsequently increased ZEB1 expression and ultimately promoted EMT and proliferation of bladder cancer cells.We demonstrated that ALKBH5 serves a critical role in bladder cancer by miRNA-mediated gene regulation,which can serve as a potential therapeutic target for this disease.