The Mechanism Of CircPTK2-PABPC1-SETDB1 Axis In Regulating Bladder Cancer Metastasis And Gemcitabine Resistance

Objective: Bladder cancer(BCa),characterized by high invasion,metastasis,recurrence,and chemoresistance,is the most common urologic malignant tumor in China.Recently,increasing studies have highlighted the significant impact of the circRNAs-protein complex in tumorigenesis and chemoresistance.However,the potential role of circRNAs and circr RNAs-protein complexes in BCa metastasis and Gemcitabine(GEM)resistance still warrant further research.Therefore,this study aims to investigate the unique role and function of circPTK2 in BCa,and further elucidate the regulatory effects of the circPTK2-PABPC1-SETDB1 axis on tumor metastasis and GEM resistance.Methods: The differentially expressed circRNAs were screened by analyzing the expression profile of circRNAs in BCa tissue.CircPTK2 was identified and characterized by Sanger sequencing,RNase R digestion,and actinomycin D treatment experiment.After circPTK2 was silenced in UM-UC-3 and T24 cells,the transcriptome was analyzed to identify target genes.Upregulated circPTK2 and SETDB1 were tested and verified in BCa tissues and cell lines.The regulatory effects of circPTK2 on SETDB1 and downstream signaling pathways were verified by biochemical experiments at the cellular and molecular levels.A circPTK2/PABPC1 interaction complex was identified by circRNA pull-down assay,mass spectrometry and bioinformatics analysis,and further confirmed by RNAbinding protein immunoprecipitation(RIP)and fluorescence co-localization;their effects on SETDB1 m RNA stability were validated by RNA decay assay.Gene set enrichment analysis(GSEA)was performed to examine the possible pathogenesis and drug resistance mechanisms of SETDB1 in BCa.In vitro functional experiments,including transwell assay and CCK-8,were used to examine the biological effect of circPTK2 and SETDB1 in tumor migration,invasion,and GEM resistance.Furthermore,a popliteal lymph node metastasis model of nude mice was established.Immunohistochemistry and HE analysis were utilized to evaluate the function of circPTK2 in vivo.Results: circPTK2 and SETDB1 were upregulated in BCa tissues,tumor cell lines,and GEM-resistant cells.Transcriptome analyses and biochemical experiments revealed SETDB1 was the downstream regulatory target of circPTK2.Functional experiments showed that the circPTK2-SETDB1 axis accelerates BCa cell migration and invasion in vitro.Importantly,using circRNA pulldown,mass spectrometry,RIP,and bioinformatic assays,we identified PABPC1 as a robust novel interacting protein of circPTK2.Mechanistically,circPTK2 could bind to PABPC1 and enhance its ability to stabilize SETDB1 m RNA,thereby specifically promoting SETDB1 expression and facilitating Wnt/β-catenin signaling pathway-mediated epithelial-mesenchymal transition(EMT).GSEA indicated that SETDB1 was highly correlated with DNA repair,homologous recombination,and multiple drug resistance in BCa.Functional experiments proved that the circPTK2-SETDB1 axis induced GEM resistance in BCa cells,while silencing circPTK2 and SETDB1 abolished these effects in vitro.Xenograft assays confirmed that circPTK2 could promote SETDB1 expression and popliteal lymph node metastasis in vivo.Conclusions: CircPTK2 functions as a significant oncogene in BCa by driving tumor metastasis and GEM resistance.Mechanistically,circPTK2 could bind to PABPC1 to form a circRNA-protein complex,thus promoting EMT and aggravating BCa malignancy by maintaining SETDB1 m RNA stability.This study suggests that the circPTK2-PABPC1-SETDB1 axis plays an important role in BCa metastasis and might serve as a novel target for treating chemoresistance in BCa.