The Study On The Regulatory Network Of Transcription Factor AP-2?

Bladder cancer is the most common malignant tumor of urinary system.For the treatment of patients with bladder cancer,surgery is commonly used in combination with chemotherapy or radiatherapy when necessary.However,the effect of treatment is not still satisfying owing to lacking of knowledge about the pathogenesis of bladder cancer.Many studies have shown that transcription factor AP-2? is a tumor suppressor,and the expression level of AP-2? is positively associated with chemotherapy sensitivity in bladder cancer,breast cancer and other cancers.In this study,the role,upstream and downstream regulation network of AP-2? in bladder cancer were systematically studied.AP-2? mainly functions as a transcription factor.In order to investigate the downstream target genes of AP-2?,we performed RNA-seq on the wildtype and AP-2?-overexpressed bladder cancer cell UM-UC-3.A total of 247 differentially expressed genes were identified,including 27 up-regulated and 220 down-regulated genes,and systemic bioinformatics analysis was proceeded to analyze their roles in bladder cancer.Four potential AP-2? targets were further confirmed with RT-PCR,one was up-regulated and three down-regulated.This result was consistent with the result of RNA-seq.In order to study the transcription regulation of AP-2? on mi RNAs,small RNA sequencing was performed on wildtype and AP-2?-overexpressed bladder cancer cell line UM-UC-3.A total of 41 differentially expressed mi RNAs were identified,including three up-regulated and 38 down-regulated genes.The function of AP-2? is influenced by protein-protein interaction.In order to investigate novel AP-2? partners,co-immuneprecipitation combined with mass spectrometry techniques were performed.The normal bladder epithelial cell line SV-HUC-1 with high expression of AP-2? was cross-linked with DSP,then immuneprecipitated with anti-AP-2? antibody or control anti-Ig G.The differential bands were subjected to mass spectrum analysis.A total of 118 potential AP-2?-interacting proteins were found.Fluorescence immunoassay was used to verify the interaction between AP-2? and Ran,one of the 118 potential AP-2?-interacting proteins,and demonstrated that AP-2? indeed interacted with Ran.In order to explore the regulation of mi RNA on AP-2? gene,bioinformatics analysis was used to screen the mi RNAs targeting AP-2?gene.The result showed that mi R-193a-5p target the coding sequence of AP-2? gene.Further study showed that knock-down of mi R-193a-5p in UM-UC-3 cells increased the expression level of AP-2? protein,while overexpression of mi R-193a-5p in SV-HUC-1 cells decreased the AP-2?expression.Moreover,mi R-193a-5p induced cisplatin resistance byinhibiting the expression of AP-2? in bladder cancer cells.In this study,the genes and mi RNAs regulated by AP-2?,AP-2?interacting partners,and the mi RNAs regulating AP-2? were systematically studied.This research deepened our understanding on the function of AP-2?,and laid a foundation for revealing the pathogenic molecular mechanism of bladder cancer and looking for new and available biomarkers.