The Effect And Mechanism Of C-MYC Targeting ODC1 And SRM Regulatory Network On Regulating Multidrug Resistance In Bladder Cancer Perfusion Chemotherapy

Objective:Bladder cancer,as one of the most frequent tumors in urinary system,has a high recurrence rate after treatment,which brings heavy mental and economic burden to patients.Non-muscular invasive bladder cancer accounts for 70%of primary bladder tumors,and the main treatment for it is operation combined with assisted intravesical perfusion chemotherapy.Early postoperative and maintenance bladder infusion chemotherapy can reduce tumor recurrence rate by killing tumor cells remaining on the wound surface.However,primary or acquired drug resistance in bladder cancer often leads to poor efficacy of chemotherapy,and even the phenomenon of multidrug resistance（MDR）after long-term use of a certain chemotherapy drug,resulting in tumor recurrence and poor prognosis of patients.Therefore,it is very important to fully understand the mechanism of multidrug resistance in bladder cancer and to find relevant targets to reverse drug resistance.Therefore,in this study,we used human bladder cancer cell line to construct a multidrug resistant cell model by a long-term and repeated administration of perfusion chemotherapy drugs.After its drug resistance was fully determined,we subsequently utilized untargeted metabolomics and transcriptomics to obtain differential metabolites and differentially expressed genes（DEGs）related to drug resistance.Afterwards,we examined their effects on drug resistance and tried to reveal the possible upstream or downstream regulatory networks.In this way,we try to reveal the specific underlying biological effects of these key factors in the development of chemotherapy tolerance and provide new theoretical strategies for reversing multidrug resistance of bladder cancer.Methods:1.Construction of multidrug resistant cell lines with chemotherapeutic drug administration:Human bladder cancer line T24 was selected as the parent cell and cultured in the medium containing pirarubicin（THP）for 24 hours.Once the state and proliferation of cells recovered after the withdrawal of drug,we repeated the chemotherapy agent administration with progressively higher concentrations of THP.CCK-8 and colony formation experiments were used detect the cell viability after drug administration.Flow cytometry and fluorescence microscope were used to detect the degree of intracellular chemotherapeutic drug accumulation after chemotherapy treatment.Thus,it was tested whether the constructed multidrug resistant cell model T24/THP developed relatively stable chemotherapy resistance.In addition,CCK-8 assay was also used to detect the cell proliferation rate,western blot was used to detect proliferation-related proteins in resistant cell lines and flow cytometry was used to detect cell cycle.In this way,other biological characteristics such as proliferation rate and cycle status of resistant cell lines were fully displayed.2.Untargeted metabolomics and transcriptome technology were utilized to find differential metabolites and differential expressed genes in multidrug resistant cell line:After extraction of metabolites,LC-MS/MS analysis was performed using a UHPLC system and mass spectrometer,and the collected metabolites data were annotated after comparison with the internal database.The variable importance projection（VIP）value for the（O）PLS model of≥1 and the P value for the t-test for univariate analysis<0.05 was used as the criteria for screening differential metabolites.KEGG pathway enrichment analysis using the R package was then performed after the differential metabolites were determined.Total RNA from resistant versus parental cells were extracted and processed into biotin-labeled c DNA.Acquisition of cellular transcriptome data was performed using Affymetrix Human Transcriptome Array 2.0 microarrays.After the raw data was processed,differentially expressed genes（DEGs）were screened using the following criteria:fold change（FC）of>2 or<0.5 and a P value of<0.05.3.Expression of polyamine metabolism-related genes in multidrug resistant cell lines:In combination with the results of metabolomics and transcriptomics,quantitative real-time PCR was used to examine the selected differentially expressed genes related to polyamine metabolism,and ornithine decarboxylase（ODC1）and spermidine synthase（SRM）were preliminarily selected as the key genes for subsequent studies.After extraction of total cellular protein,western blot experiments were used to examine whether these two enzymes were differentially expressed at the protein level between resistant and sensitive cell lines.4.Lentiviral transfection technology to verify the biological function of ODC1 and SRM on bladder cancer cell resistance phenotype in vitro and in vivo:Using lentiviral technology,ODC1and SRM were stably knocked down in human bladder cancer cell lines T24 and UMUC3,and ODC1 and SRM were stably overexpressed in the constructed multidrug resistant cell line T24/THP.First,PCR and western blot experiments were used to confirm the efficiency of transfection,and then CCK-8 drug sensitivity tests were performed to detect the alterations in chemotherapy resistance after above treatment.UMUC3 cells whose ODC1 and SRM were stably knocked down were injected into Balb/C nude mice at 4-6 weeks.Once 50 mm³ tumors were formed,chemotherapeutic drug THP was injected in situ twice a week for 2 weeks along with the observation of transplanted tumor model.The mice were sacrificed at 3 weeks of feeding and the tumor size and weight of different groups were observed.5.Search for the possible upstream of ODC1 and SRM and detect their roles in drug resistance phenotypes:The public database JASPAR was used to search for transcription factors with potential regulation effects on ODC1 and SRM,and the GEPIA was used to detect whether there existed a correlation between their expression in bladder cancer.PCR,western blot and chromatin immunoprecipitation（Ch IP）experiments were performed to verify whether these factors were differentially expressed in resistant cell lines and parental cell lines and whether they affected ODC1 and SRM expression in the cell lines we used.After the transcription factors were knocked down by transient si RNA transfection,the role they play in multidrug resistance was then detected and determined by CCK-8 drug sensitivity assay.Results:1.After treating T24 cells with THP chemotherapy with gradually increasing concentration for about 6 months,according to the median lethal concentration(IC₅₀)data and dose-survival curve,the selected multidrug resistant cell line T24/THP not only developed stable resistance to THP,but also showed tolerance to other perfusion chemotherapeutic drugs such as epirubicin（EPI）and mitomycin C（MMC）.Both fluorescence microscopy and flow cytometry results showed that under the condition of the same drug concentration and the same administration time,less accumulation of chemotherapeutic agents were detected in the resistant cell line.In addition,the resistant cell line we constructed were also characterized by slow proliferation and cell cycle arrest similar to the dormant state.2.（O）PLS-DA analysis based on the results of untargeted metabolomics revealed that there existed a significant differentiation between resistant cells and parental cells at the metabolic level.In the positive ion mode,there were 115 up-regulated and 199 down-regulated differential metabolites in the drug resistant group.In negative ion mode,94 up-regulated metabolites and 190down-regulated metabolites were detected.The results of KEGG pathway enrichment showed that the differential metabolites were most significantly enriched in arginine and proline metabolic pathway.Interestingly,in this pathway,the levels of two important polyamines,putrescine and spermidine,were significantly reduced.From the transcriptomics results,we screened 1478 up-regulated genes and 1303 down-regulated genes in the resistant cell lines.Among them,a variety of genes involved in polyamine synthesis including ODC1 and SRM were downregulated.3.Real-time PCR was used to detect genes related to polyamine metabolism.From the results obtained from RNA samples of multiple batches,it was found that ODC1 and SRM expression always tended to decrease in resistant cell line compared with the parental cell.Western blot results showed that both enzymes were significantly downregulated at protein level as well.4.Using lentiviral transfection technology,T24 cell lines with stable knockdown of ODC1And SRM were constructed.Results of PCR and western blot fully showed that expression level of two enzymes decreased in the knockdown groups.CCK-8 drug sensitivity results showed that the drug sensitivity of T24 cells to THP and EPI was increased after ODC1 and SRM knockdown.Similar experiments were also conducted using UMUC3 with stable knockdown of ODC1 and SRM and T24/THP with stable overexpression of ODC1 and SRM.The results also showed that the higher the expression levels of these two enzymes,the stronger the resistance of cells to chemotherapy occurred.In animal models,the weight of tumor formation in the ODC1and SRM knockdown groups also showed a decreasing trend.5.According to JASPAR database,we mined that c-MYC may play the role of transcription factor and bind to ODC1 and SRM promoters.Results from GEPIA website showed that there was a strong positive correlation between c-MYC content and ODC1 and SRM expression in bladder cancer.Ch IP assay confirmed that c-MYC could bind to promoters of ODC1 and SRM.Knockdown of c-MYC using si RNA transient transfection in both T24 and T24/THP also decreased the content of ODC1 and SRM.Notably,under higher THP concentrations,c-MYC levels in T24 cells did not change significantly,while in T24/THP c-MYC was markedly down-regulated.Besides,the expression level of C-MYC in T24/THP cell line was always and consistently lower than that of parental cell T24 observed in variable batches of samples.These results suggested that down-regulation of c-MYC in resistant cell models may play some unique functions.The results of the CCK-8 assay also showed that knockdown of c-MYC using two independent si RNAs in T24/THP caused an improvement in drug susceptibility of cells.At the same time,knockdown of c-MYC could cause the cell cycle arrest in G0/G1 phase,which may be related to the cell dormant state.This state may be an important reason for the formation of cancer cell multidrug resistance.Conclusion:Two key enzymes of polyamine synthesis,ODC1 and SRM,play a role in promoting the development of drug resistance in bladder cancer perfusion chemotherapy,and attenuating the expression of these two enzymes can reverse drug resistance to a certain extent.However,given that these two enzymes and polyamine themselves presented a low level in the MDR cell model,which is contradictory to their biological functions,they may not be suitable as good biological markers to reflect chemotherapy responsiveness and predict drug resistance of tumor.The decreased expression of ODC1and SRM may be related to the downregulation of c-MYC in multidrug resistant cells,while the decline of c-MYC may cause cells to enter a slowly cycling dormant state to induce the generation of resistant phenotype.