Preliminary Screening Of Biomarkers For Bladder Cancer And Chemotherapy-sensitive Bladder Cancer

Background and ObjectiveBladder cancer is the ninth most common malignancy in the world and the most common malignancy in the urinary system.Epidemiological investigations have found that bladder cancer is one of the tumors with the heaviest economic burden,mainly reflected in invasive follow-up,re-electrotomy after recurrence,controversy over treatment options for HGT1,chemotherapy resistance,and postoperative complications after radical cystectomy,etc.Therefore,it is particularly urgent to study the pathogenesis,progression and mechanism of drug resistance and to carry out clinical transformation.HGT1 refers to the invasion of subepithelial connective tissue by bladder cancer cells,and the pathological grade is high,with a 5-year muscular infiltration rate of 20%-48%.There is a controversy in the preferred treatment for HGT1 patients between bladder preservation and radical cystectomy for the past few years.In particular,there are few studies on prognosis of HGT1 bladder cancer treated by radical cystectomy,therefore,in the second chapter of this paper,we retrospectively collected the clinical data of HGT1patients undergoing radical cystectomy,and analyzed the prognostic factors of HGT1bladder cancer treated by radical cystectomy,and considered clinically treatment strategies for HGT1.Failure to identify low-risk cancer subsets often leads to overtreatment in cancer patients,however,prognostic biomarkers may improve the ability to identify risks and predict whether a patient’s cancer will return after surgery.Therefore,it is urgent to find new and reliable biomarkers to predict the efficacy and prognosis of patients effectively and accurately.EZH2 is overexpressed in many cancer types,which is considered a biomarker of poor prognosis.At present,the relationship between EZH2 expression and aggressiveness and prognosis of bladder cancer is unclear and has not been agreed.Therefore,the third chapter of this paper discussed the differences in EZH2 levels between tumors and benign tissues in patients with bladder cancer,and analyzed whether EZH2 may be a valuable biomarker associated with aggressiveness and prognosis of bladder cancer.In recent years,it has been a research trend to explore the combination of different biomarkers for subtypes classification of tumors in clinical risk assessment.At present,several molecular classification systems of bladder cancer had been proposed based on transcriptomics,among which UNC classification divided the muscle-invasive bladder cancer into luminal type and basal type.The molecular characteristics of the two groups reflected the different stages of urothelial differentiation,in which the luminal type expressed biomarkers of highly differentiated umbrella cells,such as CK20;the basal type expressed biomarkers of poorly differentiated basal cells,such as CK5 and CD44.Previous subtypes classification studies were based on transcriptome sequencing.Therefore,in the fourth chapter of this paper,the expression of CK5,CK20 and CD44 in bladder cancer were detected by tissue microarray and immunohistochemistry,and the relationship between the expression of CK5 and CK20 and the clinicopathological features and prognosis was discussed to identify subtypes of bladder cancer.The standard treatment for muscle-invasive bladder cancer is radical cystectomy and platinum-based neoadjuvant chemotherapy or adjuvant chemotherapy is selected to reduce tumor size and the probability of tumor recurrence or distant metastasis.However,resistance to platinum chemotherapy has become a difficult problem for the clinical treatment of bladder cancer and the prognosis of patients,so it is particularly important to find relevant biomarkers sensitive to chemotherapy of bladder cancer.Basic studies found that the platinum based chemotherapy sensitivity closely associated with DNA repair mechanisms.Therefore,in the fifth chapter of this paper,cisplatin resistant bladder cancer cell lines were cultured to screen biomarkers related to chemotherapy resistance of bladder cancer through DNA repair PCR array,and the value of drug resistance biomarkers in the diagnosis and treatment of patients with chemotherapy of bladder cancer was further discussed.Methods(1)90 patients with HGT1 bladder cancer were retrospectively collected.Kaplan-Meier and Log-Rank method were used for survival analysis,and univariate Cox regression analysis and multivariate Cox regression analysis were used to screen out the factors affecting the prognosis of the patients.(2)189 patients with bladder cancer were retrospectively collected,and the expression of EZH2 in bladder cancer was detected by immunohistochemistry,and the relationship between the expression level of EZH2 and the clinicopathological features and prognosis of patients was analyzed.(3)149 postoperative specimens of bladder cancer were retrospectively collected,and the expressions of CK5,CK20 and CD44 in bladder cancer were detected by tissue microarray and immunohistochemistry,and the relationship between the combination expression of CK5 and CK20 and the clinicopathologic features and prognosis of bladder cancer was discussed to identify the subtypes of bladder cancer.(4)Cisplatin resistant cell lines T24/DDP and UMUC3/DDP of T24 and UMUC3bladder cancer cells were constructed respectively,and the resistant genes of DNA repair system were screened out from T24/DDP and UMUC3/DDP by PCR array of DNA repair pathway.Results(1)Age and positive lymph node were independent risk factors of RFS,CSS and OS in patients with HGT1 treated with radical cystectomy.HGB was the independent protective factor of RFS,CSS and OS.Maximum tumor diameter≥3cm was the independent risk factor of OS.(2)EZH2 expression in bladder cancer tissues was significantly higher than that in normal urothelium.EZH2 expression level increased synchronously with tumor stage,grade and positive lymph node.Kaplan-Meier analysis showed that patients in the group with high EZH2 expression had significantly lower CSS and OS than those in the group with low EZH2 expression.Multivariate Cox regression results indicated that EZH2 protein expression was not significantly correlated with CSS and OS,while positive lymph node was an independent risk factor for CSS and OS.(3)CK5~+CK20~+subtype,CK5~+CK20~-subtype,CK5~-CK20~+subtype and CK5~-CK20~-subtype were defined by combining CK5 and CK20 expressions.Kaplan-Meier results showed that the prognosis of CK5~-CK20~+subtype was worse than other three subtypes.The independent risk factors affecting PFS,CSS and OS in bladder cancer patients were pathological T stage(muscular infiltration),positive lymph node and CK5~-CK20~+group.However,the four subtypes in the chemotherapy patient group had not yet shown significant differences in prognosis.(4)T24/DDP cell line and UMUC3/DDP cell line were successfully constructed;The resistance index of T24/DDP cell line was approximately 8,and UMUC3/DDP cell line was approximately 10.Four differentially expressed up-regulated genes were screened out between T24 and T24/DDP cell lines according to the screening criteria,including APEX1,XRCC1,FANCG and PNKP.No differentially expressed genes were screened between UMUC3 and UMUC3/DDP cell lines according to screening criteria.ConclusionsThis paper first discussed the prognostic factors of HGT1 bladder cancer patients undergoing radical cystectomy and concluded that age and positive lymph node were independent risk factors of RFS,CSS and OS,HGB was the independent protective factor of RFS,CSS and OS,and maximum tumor diameter≥3cm was the independent risk factor of OS.Due to the heterogeneity of HGT1 bladder cancer,individual diagnosis and treatment cannot be completed by histological classification alone,so we tried to study the molecular classification of bladder cancer at the molecular level.Considering that the tissue samples of HGT1 patients were insufficient for further study,so we extended our study to screen biomarkers for bladder cancer and chemotherapy-sensitive bladder cancer in combination with clinical practice.We selected EZH2,CK5 and CK20 as candidate biomarkers based on previous work and related literature reports.The high expression of EZH2 in bladder cancer was closely related to the biological behavior and poor prognosis of invasive tumors,but independent prognostic information could not be provided.The combination of CK5 and CK20 expression can identify the subtypes of bladder cancer,among which CK5~-CK20~+subtype had the worst prognosis,however,there was no significant difference in the group of chemotherapy patients,considering that the sample was too small.To further screen platinum-based chemotherapeutic resistance biomarkers for bladder cancer,we used T24 bladder cancer cell line and UMUC3 bladder cancer cell line to construct T24/DDP cell line and UMUC3/DDP cell line of cisplatin resistance respectively,and screened out differentially expressed genes through PCR array of DNA repair pathways.These findings lay the foundation for further research on the role of DNA repair genes in chemotherapy resistance of bladder cancer.