Role And Mechanism Of ADAM12 In Gemcitabine Resistance Of Bladder Cancer

Objective: Bladder cancer(BC)is one of the most common malignancies of the urinary system.Gemcitabine(GEM)is commonly used in BC.However,drug resistance may occur and result in treatment failure and disease progression.A disintegrin and metalloprotease 12(ADAM12)plays a key role in the progression of many tumors,but whether ADAM12 is involved in chemotherapy resistance of BC is still unclear.Our previous study showed that ADAM12 expression was increased in GEM resistant BC cells.Therefore,it is necessary to explore the role and mechanism of ADAM12 in GEM resistance of BC,so as to provide a new theoretical basis for the treatment of BC patients.Methods: BC patients’ transcriptome data and corresponding clinical information were download from The Cancer Genome Atlas(TCGA)database and Gene Expression Omnibus(GEO)database.Then the relationship between ADAM12 and BC patients’ TNM staging and prognosis was analyzed.In this study,BC cell lines T24 and UM-UC-3 were cultured with increased concentrations of GEM to establish GEM-resistant BC cell lines T24 GR and UM-UC-3GR.After that,PCR and Western blot were used to analyze the expression difference of ADAM12 between GEM-resistant cell lines and parental cell lines.GEM-resistant cell lines were transfected with sh RNA lentivirus to establish ADAM12 knockdown stable cell lines.Then the changes in drug resistance of cell lines after ADAM12 knockdown was analyzed.Gene set enrichment analysis,PCR,Western blot,and Cell Counting Kit-8were used to investigate the possible pathways activated by ADAM12 and the effect of combined use of pathway inhibitors and GEM.The expression of epithelial-mesenchymal transition(EMT)related markers E-cadherin,N-cadherin,Vimentin were detected in resistant cell lines and parental cell lines,and the relationship between ADAM12 and these markers was analyzed.Finally,xenograft mouse model was used to investigate the role of ADAM12 in cell migration,invasion,GEM resistance,and angiogenesis in vivo.Results: TCGA and GEO databases showed that the expression of ADAM12 was upregulated in BC tissues.The expression of ADAM12 was positively correlated with T and N stages of BC.High expression of ADAM12 indicates a poor prognosis for BC patients.GEM-resistant BC cell lines were successfully established and further studies found that ADAM12 was also upregulated.ADAM12 knockdown significantly reduced the IC50 value of GEM-resistant BC cell lines to GEM,and reversed the resistance of resistant cell lines to GEM.In addition,high expression of ADAM12 was closely related to the activation of epiderma growth factor receptor(EGFR)signaling pathway by gene enrichment analysis.Further experiments showed that ADAM12 promoted BC resistance to GEM by activating EGFR signaling pathway.The use of EGFR inhibitors also reversed the resistance of resistant cell lines to GEM.After detecting EMT-related markers,it was found that ADAM12 could also induce EMT process in BC,and promote the migration and invasion of BC cells.Finally,the role of ADAM12 in GEM resistance and EMT process in vivo was investigated,and it was found that ADAM12 also promoted angiogenesis.Conclusion: High ADAM12 expression was related to the high stage and poor prognosis of BC patients.ADAM12 was a key gene in the process of BC resistance to GEM.ADAM12 promoted BC resistance to GEM by activating the EGFR signaling pathway.For BC patients,ADAM12 might become a new valuable treatment target,and GEM combined with EGFR inhibitors might also become a new treatment strategy.