| Objective: Bladder cancer(BCa)is the most common tumor in the human urinary system.Clinically,the most common treatment method for bladder cancer is surgery combined with postoperative intravesical chemotherapy,but the recurrence rate after drug treatment is high,and after recurrence,the tumor is also resistant to other chemotherapy drugs,resulting in poor prognosis of patients.This has caused troubles to the patient’s family’s economic and mental conditions,and become an urgent issue to be solved in the current clinical treatment of bladder cancer.Multi-drug resistance(MDR)of tumors is one of the research hotspots in the medical field in recent years.Many key factors and pathways that cause tumor MDR have been discovered.However,mechanisms related to MDR in BCa are very rare.Therefore,it is the primary goal of this study to clarify the pathogenesis of bladder cancer MDR,and to find intervention methods to reduce the recurrence rate after tumor treatment and improve the prognosis of patients.Methods:1.Screening,construction,and identification of multidrug-resistant bladder cancer cell linesTwo common bladder tumor cell lines were selected as common sensitive cell lines,and the corresponding bladder cancer multidrug-resistant cell lines were gradually induced,screened,and cultured by high-dose and low-dose drug shock methods,respectively.And the cells with the optimum culture effect were selected to conduct subsequent experiments.To verify the efficiency of constructed multidrug-resistant cell line,the half-inhibitory concentration(50% maximum inhibitory concentration,IC50)of the cell line against three common clinical chemotherapeutics were determined after the multidrugresistant cell line were stable to identify the differences in cellular multidrug resistance betwwen the constructed multidrug-resistant cell line compared with the bladder cancer common sensitive cell line.2.Transcriptomic microarray sequencing combined with clinical case database information analysis to screen and identify differential genesAffymetrix HTA 2.0 Array was used in transcriptomic microarray analysis of bladder cancer multidrug-resistant cell lines and common sensitive cell lines.The 20 genes with the most significant differences were screened out according to the fold change expression levels as candidate differential genes to participate in subsequent screening and research.Through the online public clinical case database such as ONCOMINE,the expression of 10 up-regulated candidate differential genes in bladder tumor and normal bladder tissue,and their impact on the recurrence rate and progression of bladder tumor were calculated.Two factors with the most significant expression difference and correlation with bladder cancer recurrence rate were screened out by statistical analysis for the follow-up research purpose.3.Bioinformatics analysis on the expression and role of target factor in pan-cancer Combined with clinical case database data such as TCGA,the expression of the two key factors were measured in a wide range of cancer tissues and tumor cell lines,and their effects on the prognosis,immune infiltration,DNA repair and modification of various types of tumor samples were also listed.Statistics and visualization further clarify the function of the two target factors in the human body and lay the foundation for their role in bladder tumors.4.Verify the expression of key factors and their effects on the cell properties of multidrug-resistant cell linesImmunoblotting experiment was conducted to verify whether the difference in the expression level of two target factors between T24 and T24-THP was consistent with the sequencing results;then,the two target factors in the multidrug-resistant cell line were knocked down by si RNA transfection.Then,the multidrug resistance of common drug-resistant cell lines was determined to verify the effect of target factor expression changes on the multi-drug resistance of multi-drug-resistant cell lines and the factor with more obvious effects was selected for follow-up mechanism research.5.In vivo and in vitro experiments to explore specific mechanism of the target factor on the multidrug resistance of bladder cancer cellsWe constructed a stable transfected cell line of a common sensitive cell line of bladder cancer to interfere with the expression level of the target factor.After verifying the transfection efficiency,the effect of the changes in the expression level of the target factor on multidrug resistance of cells,apoptosis and related key factors was evaluated were detected and then compared with the results in multidrug-resistant cell lines.Through nude mice xenograft tumor formation experiments,it was verified that when applied with chemotherapy drugs,the size and weight of tumor were changed with the expression levels of target factors.The interaction between the target factor and the main associated factors was verified by Co IP experiment,and the cellular sublocalization of the target factor in different kinds of cells was determined by the extraction and detection experiments of nuclear protein and cytoplasmic protein and combined with drug cytotoxicity Determination experiments to explore the specific mechanism of the target factor on the multidrug resistance of bladder cancer cells.Result:1.Through selecting two stable common bladder tumor cell lines as common sensitive cell lines,the multidrug-resistant cell line of BCa(T24-THP)was cultured and constructed by low-dose drug shock method.After testing the IC50 of three drugs:Pirarubicin(THP),Epirubicin(EPI),and Mitomycin c(MMC),the results showed that the bladder cancer multidrug-resistant cell line has stronger multidrug resistance compared with common bladder cancer sensitive cell lines.2.The analysis of transcriptomic chip sequencing results suggested that 10 differential genes were significantly up-regulated in these two cell lines: RSAD2,HERC5,HERC6,HIST1H2 AC,STAT1,IFIT1,MX1,IFIT2,IFI6,and XAF1.3.Combined with clinical case databases such as TCGA and ONCOMINE,2 key genes were screened out of the 10 differentially up-regulated genes: XAF1(X chromosome-linked inhibitor of apoptosis protein associated factor 1,X chromosomelinked inhibitor of apoptosis protein associated factor 1).factor 1),IFI6(interferon alpha inducible protein 6,interferon alpha inducible protein 6).The results showed that IFI6 and XAF1 were closely related to the recurrence and progression of bladder cancer.And through pan-cancer analysis,we found the expression levels of XAF1 and IFI6 were increased in various tumor tissues and cells,and they had effects on cell phenotypes and biological activities such as cancer prognosis,immunity,DNA repair and modification.Then,the expression of the two factors and their effects on cell multidrug resistance were verified by si RNA transfection,and the gene XAF1 which showed more obvious effect was finally determined as the target gene to participate in the follow-up experiments.4.In vitro experiments and in vivo tumorigenic experiments proved that in bladder cancer common sensitive cell lines T24 and UMUC3,the changes in the expression level of the target factor XAF1 altered the cell apoptosis and cell multidrug resistance,with no difference in T24-THP cell line.The key effector proteins related to apoptosis in cells were consistent with the trend of multidrug resistance,indicating that XAF1 affects the multidrug resistance of bladder cancer cells by regulating apoptosis-related factors.5.Through Co IP immunoprecipitation and cytoplasmic and nuclear protein extraction and detection experiments,we found that in T24 cells,XAF1 was located in the nucleus and in T24-THP cells,XAF1 was located in the cytoplasm.XAF1 bound to the key apoptosis factor XIAP(X chromosome-linked inhibitor of apoptosis protein,X chromosome-linked inhibitor of apoptosis protein).The high expression of XAF1 in the nucleus caused more XIAP to accumulate in the nucleus,making it unable to exert its inhibitory effect on apoptosis in the cytoplasm,resulting in cell resistance and apoptosis;the high expression of XAF1 in the cytoplasm did not change the abundance of XIAP,thus its apoptosis inhibitory effect was not affected.When the expression of XAF1 in the cytoplasm was significantly decreased in T24-THP cell lines,the localization of XIAP was redistributed due to the change of the nuclearcytoplasmic ratio of XAF1 expression,which increased the distribution of XIAP in the nucleus and decreased the distribution of XIAP in the cytoplasm,leading to cell apoptosis and multidrug resistance.6.When treating with chemotherapeutic drugs,the endogenous expression of XAF1 in the nucleus of T24 cells increases in a short time,and promotes the entry of XIAP into the nucleus,resulting in apoptosis and making the cells non-resistant,while T24-THP cells could resist in a short time.The endogenous expression of XAF1 induced by chemotherapeutic drugs was increased,resulting in multidrug resistance.Conclusion: XAF1 is a common apoptosis-related protein in tumor cells and an important related factor of XIAP,which can antagonize the anti-apoptotic effect of XIAP.XAF1 was significantly up-regulated in multidrug-resistant bladder cell lines compared with common cell lines.The trend of cell multidrug resistance tiggered by the changes of XAF1 expression levels in these two cell lines was completely opposite to that in cell apoptosis.Long-term drug induction contributo to the shuttle of XAF1 from the nucleus to the cytoplasm in multidrug-resistant cell lines,thereby leading to the distribution of XIAP expression in cells and reducing apoptosis and promoting cell multidrug resistance.Multidrug-resistant cell lines could also resist the elevated endogenous expression of XAF1 caused by short-term chemotherapeutics,thus rendering the cells resistance. |
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