Research On The Mechanism Of Cisplatin Resistance Induced By CircUGGT2 In Bladder Cancer Through Nonhomologous End-joining Pathway

OBJECTIVE: Bladder cancer ranks among the most prevalent urological malignancies,and a cisplatin(CDDP)-based chemotherapy regimen is the standard of treatment for locally advanced and metastatic bladder cancer.However,the prognosis for bladder cancer patients remains unsatisfactory,and one of the most significant reasons is the resistance of bladder cancer to CDDP.As a result,investigating the mechanism of CDDP resistance in bladder cancer has become a crucial research direction.Circular RNA(circ RNA)is a type of non-coding RNA characterized by a circular structure.In recent years,increasing evidence has demonstrated the involvement of circ RNA in cancer,including its role in promoting chemotherapy resistance.However,the underlying mechanisms of drug resistance remain largely unknown.Thus,the objective of this study was to investigate the mechanisms by which circ UGGT2 promotes CDDP resistance in bladder cancer.METHODS: In this study,we conducted a screening of highly expressed circular RNAs in bladder cancer using the GEO database,and identified circ UGGT2 as a potential target molecule.Real-time fluorescence quantitative polymerase chain reaction assays were used to verify its high expression levels in bladder cancer tissues and cell lines.We verified the circular structure of circ UGGT2 using Divergent primer assay and RNase R digestion assay.Furthermore,we determined the subcellular localization of the target molecule using nucleoplasmic separation assay and RNA fluorescence in situ hybridization.To investigate the effect of circ UGGT2 expression level on the migration and invasion ability of bladder cancer cells,we performed Scratch assay and Transwell invasion assay.To investigate the role and function of circ UGGT2,we classified the bladder cancer patients into high and low expression groups based on their circ UGGT2 expression levels,and conducted follow-up and statistical analysis.We then utilized circ RNA pull-down assays and mass spectrometry to identify the most abundant circ RNA-binding proteins,and subsequently elucidated circ UGGT2-associated pathways.We confirmed the role of the target molecule in promoting CDDP resistance in bladder cancer cells by overexpressing or knocking down circ UGGT2,as well as its downstream binding proteins,X-ray repair cross-complementary protein 6(Ku70/XRCC6)and X-ray repair cross-complementary protein 5(KU80/XRCC5).We examined the cell apoptosis response to CDDP using flow cytometry and light microscopy experiments in the presence of CDDP.This study also conducted in vivo experiments to further confirm the role of circ UGGT2 in promoting CDDP resistance in bladder cancer.To determine the specific mechanism of circ UGGT2 in the non-homologous end-joining(NHEJ)pathway,we extracted chromatin complexes for western blot(WB)assays to compare total cellular proteins and verify the mechanism of the target molecule in promoting CDDP resistance.Finally,we used immunofluorescence and WB assays to detect the level of phosphorylated histone H2AX(γH2AX),a DNA repair marker,to further demonstrate the role of circ UGGT2 in the NHEJ pathway.RESULTS: The high expression of circ UGGT2 was verified in GEO dataset GSE92675,bladder cancer tissues and cell lines.Scratch and Transwell invasion assays confirmed that circ UGGT2 promotes migration and invasion of bladder cancer cells.Furthermore,follow-up results of patients included in the study revealed worse prognosis in the circ UGGT2 high expression group.The circ RNA pull-down assay showed that circ UGGT2 binds to the KU protein,an essential DNA repair factor in the NHEJ pathway,which may be involved in CDDP resistance.In this study,we confirmed the positive correlation between circ UGGT2 and CDDP resistance in bladder cancer cells using flow cytometry and light microscopy assays.We also found that circ UGGT2 promoted CDDP resistance by binding to KU protein.Furthermore,we conducted WB assays after extracting chromatin complexes and total proteins of bladder cancer cells for comparison and found that the levels of DNA repair factors KU70,KU80,DNA-dependent protein kinase catalytic subunit(DNA-PKcs)and X-ray repair cross-complementing protein 4(XRCC4)in chromatin were reduced in circ UGGT2 knockdown bladder cancer cells under the effect of CDDP,while the levels of the above molecules in total cellular proteins were unchanged.The findings of this study indicate that circ UGGT2 promotes CDDP resistance by facilitating the recruitment of repair factors to DNA breaks through its binding to KU proteins.The level of DNA repair can be assessed by monitoring γH2AX,a marker of DNA damage response.Our investigation revealed that knockdown of circ UGGT2 led to decreased levels of γH2AX in bladder cancer cells treated with CDDP,indicating its involvement in the NHEJ pathway of DNA repair.CONCLUSIONS: In bladder cancer,circ UGGT2 is highly expressed and promotes invasion and progression,significantly affecting prognosis.Through its interaction with KU protein,a critical DNA repair factor in the NHEJ pathway,circ UGGT2 recruits DNA-PKcs,XRCC4,and other repair factors to mediate CDDP resistance in bladder cancer.