Correlation Between SLFN11 Expression And Drug Resistance Of Bladder Cancer Cells And Its Expression Regulation

SLFN11,a member of the Schlafen family,has endonuclease and helicase activities.It has been reported to be related to the sensitivity of different kinds of cancer cells to DNA-damaging agents.However,SLFN11 is rarely studied as a biomarker and drug target in bladder cancer.In this paper,we studied the correlation between the expression of SLFN11 and the drug resistance of bladder cancer cells and explored the possible reasons for the low expression of SLFN11 in bladder cancer cells.Firstly,we used the website tools to analyze the expression data of SLFN family genes in various cancers,including bladder cancer.The expression of SLFN11 in most tumor samples is lower than that in normal samples.We chose SLFN11 for further study because it seemed to be relatively more valuable in bladder cancer.At the same time,clinical parameters of bladder cancer,inluding race,weight,age,histological subtype,and molecular subtype were closely related to the expression of SLFN11.Gene ontology(GO)enrichment analysis and protein-protein interaction(PPI)analysis were applied to the top 200 similar genes of SLFN11,which predicted the cytological processes and functions that SLFN11 may involve.It was found that SLFN11 involves in DNA metabolism,mitotic cell cycle,etc.,and its functions are closely related to DNA damage response,DNA repair,etc.After that,we performed a prognostic correlation analysis of SLFN11.The results showed that patients with lower expression of SLFN11 in bladder cancer had a longer overall survival period.In addition,the expression of SLFN11 was positively correlated with the sensitivity of cancer cells to DNAdamaging drugs(such as SN-38 and gemcitabine).To verify the reliability of bioinformatics analysis,we used human bladder cancer cells as models and constructed a drug-resistant cell line T24-GR through drug induction.The drugresistant cell line was different from T24 in morphology and proliferation speed.Flow cytometry results showed that the proportion of S phase cells in T24-GR was more than that in T24 and the cycle arrest by gemcitabine was less than in T24.CCK-8 assay was used to determine the toxicity of gemcitabine to bladder cancer cells.The results of real-time quantitative PCR and Western blot assay showed that the expression of SLFN11 in the bladder cancer cells we used(UM-UC-3,T24,T24-GR)was lower than that in the normal urothelial cell(SV-HUC-1),and the order of SLFN11 expression was consistent with the order of sensitivity to gemcitabine(UM-UC-3>T24>T24-GR).Entinostat,an HDAC inhibitor,could significantly up-regulate the expression of SLFN11 in UM-UC-3 cells and increased its sensitivity to gemcitabine and two derivatives.We constructed CRISPR-Cas9 plasmids for SLFN11 knockout,SLFN11-KO1,and SLFN11-KO2.After the two plasmids were transiently transfected into UM-UC-3 cells,the expression of SLFN11 decreased and the cell cycle arrest by gemcitabine was reduced.Finally,we studied the reasons that may cause the low expression of SLFN11 in bladder cancer cells.Firstly,we used cBioPortal to analyze the genetic alterations of SLFN11 in bladder cancer cases.The results showed that the genetic alteration were not significantly correlated with the survival of the patients.We further analyzed the effect of methylation on SLFN11 expression and found that the methylation density of some CpG sites was closely related to SLFN11 expression,including positively correlated site cg18124488(located in the promoter region)and negatively correlated site cg18108623(not located in the promoter region).Through the methylation specific PCR experiment,we proved that among the bladder cancer cells,methylation of the SLFN11 promoter was detected in drug-resistant cell line T24-GR,which was a probable reason for the low expression of SLFN11 in T24-GR.In addition,we thought that ZNF75D was a possible transcription factor of SLFN11 through transcription factor prediction.The expression analysis results of qPCR showed that in UMUC-3 and T24,the expression of ZNF75D was consistent with that of SLFN11.Therefore,ZNF75D might affect the expression of SLFN11.In conclusion,the expression of SLFN11 in bladder cancer is closely related to some clinical symptoms and is positively related to the sensitivity of bladder cancer cells to DNA damage agents,such as gemcitabine.The factors that cause the difference in its expression may be methylation and transcription factor ZNF75D.SLFN11 can be used as a drug prognostic marker for bladder cancer and a therapeutic target related to the drug combination.