Mechanism Of HNF1B Involved In The Progression Of Bladder Cancer Through DNA Methylation Regulation

Background:Bladder cancer is one of the most common malignancies in genitourinary system,with an increasing trend of incidence in the past years.Muscle-invasive bladder cancer accounts for about 25%～30%of bladder cancer,which is characterized by high malignancy,rapid progression,easy metastasis and easy recurrence.While non-muscle-invasive bladder cancer accounts for approximately 70%～75%of bladder cancer.These patients remain at risk of recurrence or progression even after conventional therapy.Recently,novel treatment modalities such as immunotherapy,neoadjuvant chemotherapy and targeted therapy have shown considerable efficacy in patients with bladder cancer.However,the response rates to these treatments are suboptimal in patients with advanced bladder cancer due to the impact of drug resistance and treatment-related toxicities.Therefore,exploring effective prognostic biomarkers is helpful for risk stratification,and investigating the molecular mechanisms of bladder cancer progression helps mining novel therapeutic targets.These are the key points to achieve precision treatment for bladder cancer.Objective:To explore effective prognostic biomarker in bladder cancer;to explore the biological functions and regulatory mechanisms of HNF1B in bladder cancer;to explore the molecular mechanism by which HNF1B plays a role in bladder cancer.Methods:We constructed a bladder cancer prognostic signature based on TCGA bladder cancer data,and selected the important model gene HNF1B for in-depth study.First,this study investigated the biological function of HNF1B gene in bladder cancer at cellular,animal,and clinical levels.We estimated the relationship between HNF1B expression and clinicopathological charicteristics in our clinical cohort.We constructed HNF1B overexpression or knockdown cell models to perform cell proliferation assays,clonogenic,wound healing,migration and invasion assays to explore the effect of HNF1B on the phenotypes of bladder cancer cells.We analyzed the effect of HNF1B on tumor growth in vivo by constructing HNF1B overexpression xenograft in nude mice.Second,this study explored the regulatory mechanism of HNF1B in bladder cancer based on TCGA database.As for DNA methylation regulatory mechanism,we used bisulfite genomic sequencing PCR to confirm the methylation modification of HNF1B in bladder cancer cell lines,and found that methylation inhibitors treatment restored HNF1B expression.Pyrosequencing then demonstrated that HNF1B expression was regulated by methylation in bladder cancer tissues.Finally,we conducted transcriptome sequencing to explore the downstream signaling pathways of HNF1B in bladder cancer,and validated them at the protein level.At the same time,we combined transcriptome sequencing and ChIP sequencing to mine the target genes of HNF1B.Results:The KEGG-related prognostic signature showed good predictive efficacy for long-term outcome in bladder cancer(3-year AUC=0.76,5-year AUC=0.75).HNF1B,as one of the model genes,was significantly associated with patient prognosis,subtype,pathological grade and AJCC stage(P<0.05).HNF1B mainly plays a transcriptional regulatory role,which is involved in the development and progression of several tumors.Therefore,we deeply explored its value in bladder cancer.In bladder cancer cell lines,knockdown of HNF1B significantly promoted cell proliferation,migration and invasion;whereas overexpression of HNF1B significantly suppressed cell proliferation,migration and invasion.HNF1B overexpressing xenografts have a significantly slower growth(P<0.05).Based on TCGA database analysis,HNF1B promoter methylation level was negatively correlated with HNF1B expression(P<0.001).HNF1B promoter hypermethylation was detected in UM-UC-3 and 5637 cells,and methylation inhibitor treatment significantly upregulated mRNA and protein levels of HNF1B in the two cell lines.Consistently,pyrosequencing results showed that the methylation level of HNF1B promoter was negatively correlated with HNF1B expression in bladder cancer tissues(P<0.05),and it correlated with clinicopathological characteristics of bladder cancer patient.In bladder cancer cells,HNF1B knockdown activated the Ras/MEK/ERK pathway and EMT process,whereas overexpression inhibited them.The results of tissue microarray confirmed that HNF1B high expression inhibited the Ras/MEK/ERK pathway(P<0.05).HNF1B differentially expressed cell lines were differently sensitive to MEK inhibitor,and MEK inhibitor could effectively inhibit the hyperproliferation induced by HNF1B low expression.We identified five potential target genes downstream of HNF1B.Among them,PPFIBP2 expression was positively correlated with HNF1B(P<0.05).In addition,PPFIBP2 was strongly associated with patient prognosis as well as subtype and AJCC stage of bladder cancer(P<0.05).Conclusions:1.HNF1B expression correlates with patient prognosis,subtype,pathological grade,and AJCC stage of bladder cancer.2.HNF1B can inhibit cell proliferation,migration and invasion in bladder cancer.3.HNF1B expression is mainly regulated by DNA methylation in bladder cancer.4.HNF1B can inhibit Ras/MEK/ERK pathway and EMT process to affect bladder cancer progression.