Genome-wide Screening Identifies Genes AKR1C1 Critical For Resistance To Pirarubicin In Bladder Cancer

Background:Non-muscle-invasive bladder cancer（NMIBC）is a common tumor of the urinary system.Given its characteristics of high rates of recurrence,progression,and drug resistance,it seriously affects the quality of life and limits the survival time of patients.Pirarubicin（THP）is a bladder infusion chemotherapy drug recommended by the guidelines for NMIBC.Although the widespread use of THP reduces the recurrence rate of NMIBC,10%–50%of patients with NMIBC still experience tumor recurrence,which is closely related to tumor resistance to chemotherapy drugs,within 5 years after receiving THP infusion chemotherapy.The CRISPR/dCas9-SAM system is an improved technology based on CRISPR/Cas9.In contrast to the CRISPR/Cas9 system with target gene knockout as the mechanism of action,CRISPR/dCas9-SAM can overexpress endogenous target genes.Therefore,this technology is widely used in the research on finding the drug resistance genes of pathogenic microbes.Aldo-keto reductase family 1 member C1（AKR1C1）is a member of the aldo-keto reductase superfamily.Although it has been reported to be related to the chemoresistance of numerous tumors,it has not been reported in tumor drug resistance related to NMIBC or THP treatment.Objective:This study aims to screen the key genes causing THP resistance in bladder cancer cell lines and their mechanisms by using the CRISPR/dCas9-SAM system.The research results will provide new theoretical bases and therapeutic targets for THP resistance in bladder cancer and novel solutions for the clinical prevention and even reversal of THP resistance in bladder cancer.Methods:1.The whole-genome wild CRISPR/dCas9-SAM system was used to screen the key genes of bladder cancer cells with THP resistance,and the results were visually interpreted through bioinformatics methods to verify that AKR1C1 is a potential target gene that causes the THP resistance of bladder cancer cells.2.The expression level of AKR1C1 in bladder cancer tissues and cell lines collected clinically was detected through q RT-PCR and immunohistochemistry,and the level of AKR1C1 in the tissues of patients with initial/recurrent bladder cancer was analyzed.3.The stable overexpression transformant of T24-AKR1C1 was constructed on the basis of T24 cells via lentiviral transfection.The overexpression of AKR1C1 was verified by q RT-PCR and Western blot analysis,and its effects on the pharmacological characteristics of T24 cell lines and the proliferation,invasion,migration,and other functions of tumor cells were investigated.The effects of the AKR1C1 gene on the levels of reactive oxygen species（ROS）and 4-hydroxynonanal（4-HNE）were detected to verify that AKR1C1 causes T24 cell line to resist THP-induced apoptosis.Similarly,in RT4 bladder cancer cell lines,small interfering RNA（si RNA）or the AKR1C1 inhibitor aspirin was used to interfere with the expression of AKR1C1 or inhibit its function.The effect of AKR1C1 on the pharmacological characteristics and cellular function of bladder cancer cell lines was reversed by downregulating the expression or function of AKR1C1,and the changes in ROS and 4-HNE expression levels were detected.4.The ability of THP to induce apoptosis in the T24-AKR1C1-overexpressing cell line and RT4 AKR1C1-inhibited cell line was detected via a TUNEL test,and the expression levels of apoptosis-related proteins in the two groups of cells were verified by Western blot analysis.5.Western blot analysis confirmed that in bladder cancer cells,THP could upregulate the expression of AKR1C1 through the ROS/KEAP1/NRF2 axis,whereas the ROS inhibitor Tempol could inhibit the THP-induced upregulation of the AKR1C1 gene and prevent drug resistance.6.By using a xenograft tumor model of immunodeficient BLAB/c mice,AKR1C1 was verified in vivo to be capable of causing bladder cancer to develop THP resistance.Results:1.CRISPR/dCas9-SAM whole genome screening combined with THP-positive screening revealed that AKR1C1 was the gene with the highest enrichment and was a potential target gene that caused the resistance of bladder cancer to THP.2.AKR1C1 was expressed at lower levels in T24 cells than in RT4cells.In clinical tissues,the expression of AKR1C1 in bladder cancer tissues was significantly higher than that in adjacent tissues.Moreover,the expression of AKR1C1 in Ta-T1 bladder cancer tissues was higher than that in T2-T3 bladder cancer tissues.In addition,the expression of the AKR1C1 gene in recurrent bladder cancer was significantly higher than that in primary bladder cancer.3.In the T24 bladder cancer cell line,the lentiviral overexpression of the AKR1C1 gene can enhance the IC₅₀ of THP for cells,causing cell resistance,but will not change the proliferation,invasion,and migration abilities and other functions of tumor cells.Similarly,in RT4 cells,interfering with the expression of AKR1C1 by si RNA treatment or inhibiting the function of AKR1C1 by aspirin treatment can reduce the IC₅₀of THP for cells and enhance sensitivity to drugs and did not affect cell proliferation and other functions.4.In T24 bladder cancer cells,the overexpression of the AKR1C1gene can reduce the production of 4-HNE and the level of ROS,thereby inhibiting the ability of THP to induce apoptosis.By contrast,in RT4bladder cancer cells,inhibiting the function of AKR1C1 through inhibitors can increase the production of 4-HNE,thereby increasing the level of ROS and thus promoting the ability of THP to induce apoptosis.5.In T24 cells,THP can promote the uncoupling of KEAP1 and NRF2and promote the phosphorylation of NRF2,thereby upregulating the expression of AKR1C1 and gradually causing resistance to THP.The use of the ROS scavenger Tempol can inhibit the THP-induced upregulation of the AKR1C1 gene and prevent the emergence of drug resistance.6.In BALB/c mice with xenotransplantation immunodeficiency,the overexpression of AKR1C1 can cause tumor resistance to THP treatment in vivo.HE staining revealed that the overexpression of the AKR1C1 gene can reduce the death of tumor cells after THP treatment.Immunohistochemical staining demonstrated that the overexpression of the AKR1C1 gene will not cause changes in Ki67 expression.Conclusion:1.The expression of AKR1C1 was higher in bladder cancer tissues than in adjacent tissues,higher in non muscle invasive bladder cancer tissues than in muscle invasive bladder cancer tissues,and higher in recurrent bladder cancer tissues than in bladder cancer tissues found for the first time;2.AKR1C1 can improve the resistance of bladder cancer cells to THP and resist THP induced apoptosis in vitro,but does not affect the proliferation,migration and invasion of bladder cancer cells.Aspirin can inhibit the function of AKR1C1 and reduce the resistance of bladder cancer to THP;3.THP can up regulate the expression of AKR1C1 in bladder cancer cells through ROS/KEAP1/NRF2 axis,and Tempol can repress this signal pathway by clearing ROS;4.AKR1C1 can improve the drug resistance of bladder cancer tissue to THP in xenotransplantation mice,and reduce the cell death caused by THP treatment in cancer tissue in vivo.