Study On The Mechanism Of FBX Regulating The Drug Resistance Of Bladder Cancer Stem Cells

Bladder cancer is the most serious malignant tumor of the urinary system and its incidence is increasing year by year.In 2020,there were570,000 new diagnoses and 210,000 deaths from bladder cancer.Surgery,chemotherapy,immunotherapy and targeted therapy are the main approaches for bladder cancer treatment,but they still can’t reduce the recurrence rate of bladder cancer.Bladder cancer stem cells,also known as bladder cancer initiating cells,have surface markers such as CD44 and CD24,and maintain self-renewal through pathways such as Hedgehog.Bladder cancer stem cells are drug-resistant,and it is difficult to clear them with traditional treatment methods,which eventually leads to drug resistance and recurrence in patients.Therefore,understanding the drug resistance mechanism of bladder cancer stem cells and screening drugs targeting bladder cancer stem cells are new directions in the field of bladder cancer treatment.Previous study has shown that KMT1 A is a bladder cancer stem cell marker.Chaetocin can significantly inhibit the proliferation of bladder cancer stem cells,induce apoptosis and necrosis,arrest the cell cycle in G1 phase,and reduce tumorigenicity by targeting KMT1 A.Chaetocin can’t completely clear bladder cancer stem cells,nor reduce the size of the xenograft tumors,indicating the heterogeneity of bladder cancer stem cells.To explore the resistance molecular mechanism in bladder cancer stem cells to Chaetocin,RNA-seq and RT-PCR were performed on wild-type T24 cells and Chaetocin-resistant T24 cells.The result showed that the expression of ubiquitination-related protein FBX was significantly up-regulated in resistant cells.In order to verify the effect of FBX on the phenotype and the drug resistance of bladder cancer cells,the expression of FBX was inhibited by RNA interference.The experimental results showed that FBX knockdown could inhibit the proliferation of bladder cancer cells(inhibition rate: 9.8-30.6%),up-regulate the proportion of apoptotic and necroptotic cells(1.41-2.69 times),reduce the expression of stemness-related genes(inhibition rate: 34.1-71.5%).Under the condition of Chaetocin treatment,the proliferation rate of FBX knockdown cells decreased(inhibition rate: 17.1-34.5%),and the proportion of apoptotic and necroptotic cells was increased(1.67-2.13times).BC is a targeted inhibitor of FBX,and the combination therapy of Chaetocin and BC can more effectively inhibit the proliferation,induce apoptosis and necrosis,affect cell cycle distribution,and reduce the expression of stemness-related genes of bladder cancer cells compared with Chaetocin alone.The above results indicate that FBX gene can affect the phenotype of bladder cancer cells and lead to the resistance of bladder cancer cells to Chaetocin.Combination therapy of Chaetocin and BC more effectively clears bladder cancer cells and is a new bladder cancer treatment option.