| BackgroundGlobally,the incidence of bladder cancer is increasing year by year.75%of bladder cancer is non-muscle invasive bladder cancer（NMIBC）,30%of NMIBC will develop into muscle invasive bladder cancer（MIBC）over time,and 10%of MIBC will be diagnosed with local or distant metastasis.For MIBC,although radical cystectomy can improve the five-year survival rate of some patients,50%of patients still will experience tumor-specific death.The occurrence,local invasion and early metastasis of tumors are related to cell biological characteristics such as abnormal cell proliferation,migration and invasion,and these biological characteristics are often regulated by certain kinase signaling pathways.Therefore,the discovery and confirmation of signaling pathways related to the occurrence and development of bladder cancer and the identification of a certain molecular marker that can reflect the proliferation of bladder cancer cells,can provide an important reference for clinical molecular biological diagnosis of bladder cancer and the selection of potential therapeutic targets.Thymidine Kinase（TK）is a key enzyme or rate-limiting enzyme in the process of DNA salvage synthesis.There are two kinds of TK in mammals,namely cytoplasmic thymidine kinase（thymidine kinasel,TK1）and mitochondrial thymidine kinase（thymidine Kinase2,TK2）.TK1 in the tissues can be detected by peripheral blood after it is released into the blood,which is called Serum thymidine kinase 1（STK1）.TK1 can catalyze the phosphorylation of thymidine（thymidine deoxyribose,TdR）to thymidylate（deoxythymidine monophosphate,dTMP）,which is one of the four essential nucleotides for DNA synthesis.Existing studies had confirmed that the expression level of TK1 in breast cancer,thyroid cancer and other solid tumors was significantly increased.After studying MIBC,Steffen found that the expression of TK1 in MIBC was significantly higher than that of normal urothelium,and proposed that TK1 had potential diagnostic marker for bladder cancer.However,the specific mechanism of TK1 in the occurrence and development of bladder cancer is not yet clear.The purpose of this study was to verify the significant role of TK1 in bladder cancer and to clarify its molecular mechanism.Through the analysis of TCGA and STRING database,it was found that TK1 gene interacted with TFDP1（Transcription factor Dp-1,TFDP1）gene.TFDP1 was a transcription factor that could encode DP1 protein.Studies had confirmed that TFDP1 played an important role in breast cancer,hepatocellular carcinoma and lung cancer through the Wnt/β-catenin pathway.wnt/β-catenin is the classic Wnt signaling pathway,The Wnt signaling pathway is an evolutionarily conserved pathway related to tumorigenesis,which regulates various cellular activities such as stem cell self-renewal,differentiation and tissue homeostasis.Studies had reported that the activation of Wnt signaling pathway was closely related to the proliferation of neuroblastoma,osteosarcoma,etc.Recent studies had confirmed that the Wnt/β-catenin signaling pathway played an important role in the occurrence and development of bladder cancer.Therefore,we proposed a hypothesis that TFDP1 played a role in bladder cancer through the Wnt/β-catenin pathway,and TK1 might regulate the Wnt/β-catenin signaling pathway through TFDP1.At present,there is no relevant experimental research report.In view of this,we had studied the regulatory relationship of TK1,TFDP1 and β-catenin in bladder cancer.Part Ⅰ The expression and clinical significance of TK1 in bladder cancer ObjectiveTo explore the expression characteristics of TK1 in bladder cancer patients and healthy population,and to prove its feasibility as a marker of bladder cancer proliferation.MethodsThe subjects were 60 patients with bladder urothelial carcinoma who underwent surgical resection in the Department of Urology from January 2015 to June 2018.Another 60 healthy physical examiners from the Physical Examination Center were collected as control group.The fasting blood sample in the morning was taken 3 days before the operation,another blood samples were taken at postoperative 1 week,3 months and 6 months in BUC.And random fasting blood samples were taken from healthy control group.The concentration of Serum TK1（STK1）was detected by immunoblotting enhanced chemiluminescence method.The concentration of STK1,the number of positive cases and negative cases were recorded.The expression of TK1,Ki-67,MCM2,MTA2 and PCNA in the excised specimens were detected by immunohistochemistry.The statistical analysis of the data was carried out by SPSS21.0 and SPSS26.0 software.The counting data was expressed as percentage,and theχ2 test was used.The measurement data was expressed as mean±standard deviation（x±s）,and the paired t test or two separate sample t test was used.The two comparisons of the mean between groups were analyzed by variance analysis.The single factor repeated measurement data was analyzed by single factor repeated measurement variance analysis.P<0.05 was regarded statistically significant.Results1.The positive rate of STK1 in the BUC group was higher than that in the healthy control group（78.33%vs 21.67%,P<0.001）;The concentration of serum TK1 in BUC group was higher than that of healthy（3.08±1.51 pmol/1 vs 1.34±0.99 pmol/1,P<0.001）.2.The serum TK1 concentration of BUC patients decreased to a negative expression level in the 3rd month after a transient increase.3.The sensitivity of serum TK1 to detect BUC was 78.33%（47/60）.The ROC curve analysis showed that the area under the curve of serum TK1 detecting BUC was 0.787,and the 95%confidence interval was（0.692,0.882）.4.The expression of serum TK1 had nothing to do with BUC staging（2.65±1.65pmol/l vs 3.35±1.39pmol/l,P=0.082）and classification（3.05±1.37pmol/l vs 3.04±1.59pmol/l,P=0.981）.5.The strong positive expression rate of TK1 in tissue samples after BUC was significantly higher than Ki-67,MCM2,MTA2 and PCNA（P<0.01）.ConclusionsTK1 was highly expressed in patients with bladder cancer,which was a marker for proliferation of bladder cancer and had potential diagnostic value for bladder cancer.Part Ⅱ Bioinformatics analysis of the correlation between TK1 and TFDP1 ObjectiveTo verify the expression of TK1 in bladder cancer and adjacent tissues by bioinformatics analysis.To detect the expression of TK1 in bladder cancer cells,and to analyze the pathway and co-expression genes,so as to further explore the mechanism of TK1.Methods1.TCGA,GTEX and CCLE database data for TK1 single gene multi-dimensional extensive cancer analysis were downloaded.2.The data of bladder cancer and adjacent tissues in TCGA were analyzed by differential mRNA analysis,TK1 differential expression analysis.Meanwhile,the correlation between TK1 and staging of bladder cancer was verified.3.TK1-High up-regulated genes in TK1 differential expression were analyed by Hiplot platform,and protein interaction network was constructed.TK1 and TFDP1 gene expression correlation was analyzed by this platform.The correlation between TK1 and TFDP1 gene expression was further analyzed through this platform.4.Through the analysis of GEO and Array Express database files,the correlation between TK1 and TFDP1,the enrichment of TK1 pathway,the enrichment of pathway-related genes,and the way that TK1 participates in cell cycle regulation were verified.Results1.Extensive cancer analysis showed that TK1 was highly expressed in bladder cancer.2.Differential expression analysis of mRNA expression data in 414 cases of bladder cancer tissue samples and 19 cases of bladder cancer adjacent normal tissue samples confirmed that TK1 gene expression was upregulated in bladder cancer.3.The expression of TK1 gene in bladder cancer samples was significantly higher than that in adjacent tissues,and TK1 was stably expressed in different stages of bladder cancer.4.Differential expression analysis between TK1 high group and TK1 low group showed that TFDP1 was up-regulated in TK1 high group.PPI network analysis and correlation analysis of TFDP1 and TK1 gene expression showed that TK1 was highly correlated with TFDP1,with the Pearson correlation coefficient was 0.39,R value was 1.41,P<0.001 and 95%CI（0.3,0.47）.5.Co-expression genes analysis with 5637 and T24 bladder cancer cell lines in GEO and Array Express databases showed that TK1 and TFDP1 were coexpressed genes in bladder cancer cell lines.6.The three major enrichment pathways of bladder cancer cells were purine,pyrimidine and cell cycle pathways.TK1 was an important pyrimidine metabolism pathway related gene,and TFDP1 was an important cell cycle pathway related gene.Enrichment analysis of pathway related genes suggested that TK1 might be involved in the cell cycle regulation of bladder cancer through TFDP1.Conclusions1.The expression of TK1 had correlation and protein interaction with TFDP1.2.TK1 was an important pyrimidine metabolism related gene,and TFDP1 was an important cell cycle related gene.Part Ⅲ The effect of TK1 on the biological behavior of bladder cancer cells and the study of TK1-TFDP1-Wnt/β-catenin pathwayObjective1.To detect the difference of TK1 expression in different bladder cancer cell lines,and to verify the effect of TK1 overexpression and knockdown on proliferation,apoptosis,migration and invasion of bladder cancer cells.2.To demonstrate the relationships between TK1,TFDP1 and Wnt/β-catenin pathway in bladder cancer cell lines.Methods1.Expression of TK1 gene in different bladder cancer cell lines:Five bladder cancer cell lines T24,5637,J82,EJ and RT112 were resuscitated and cultured.RT-PCR and Western blot were used to detect the mRNA and protein expression levels of TK1 gene in the five cell lines.According to the results of preliminary experiments,we selected bladder cancer cell lines with high expression of TK1 mRNA and protein for TK1 knockdown,and selected bladder cancer cell lines with low expression of TK1 mRNA and protein for TK1 overexpression;2.TK1 gene overexpression and knockdown effect monitoring:TK1 overexpression of lentivirus was constructed to transfect bladder cancer cells.RT-PCR and Western blot were used to detect the expression of TK1 to confirm the overexpression effect.TK1 gene siRNAs were designed and synthesized,which were used to transfect bladder cancer cells.RT-PCR and Western blot were used to detect the change of TK1 expression level,and the best knockdown siRNA was selected for subsequent experiments.3.Explore the regulatory relationship between TK1,TFDP1 and β-catenin pathway:After knockdown and overexpression of TK1 and TFDP1 in bladder cancer cells,the expression of TFDP1 and β-catenin were tested.Then,the regulatory relationship between TK1,TFDP1 and β-catenin were further verified by rescue assays.4.TK1 and TFDP1 protein interaction detection:The TK1/TFDP1 complex was immunoprecipitated by Co-inmunoprecipitation technology,and the interaction between TK1 and TFDP1 protein was verified by Western blot.5.Detection of the influence of TK1 overexpression and knockdown on the biological behavior of bladder cancer cell lines:After overexpress and knockdown of TK1 in bladder cancer cells,CCK8 was used to detect the changes of cell proliferation curve.The effects of apoptosis and cell cycle were detected by flow cytometry.Transwell was used to detect the changes of cell migration and invasion.6.The effect of the interaction between TK1 and TFDP1 on the biological behavior of bladder cancer cell lines:After overexpressing TK1 and knocking down TFDP1 in bladder cancer cells,CCK8 was used to detect the changes of cell proliferation curve.The effects of apoptosis and cell cycle were detected by flow cytometry.Transwell was used to detect the changes of cell migration and invasion.7.Effects of overexpression and knockdown of TK1 on biological behavior of bladder cancer cell lines in vivo experiments:Nude mice feeding 4-6 weeks were inoculated with bladder cancer cells which had been logarithmically prolonged after TK1 overexpression and knockdown.After 5-7 days,the tumor proliferation was observed.The maximum diameter（a）and minimum diameter（b）of the tumor were recorded twice a week,then the tumor proliferation curve were made and the tumorigenesis was observed.After the experiment,the nude mice were sacrificed by decapitation,and the tumors were weighed and counted.The tumor tissue mRNA and protein were extracted to detect the expression of TK1,the tissue morphology was observed by HE staining,and the expression of TFDP1 and β-catenin were detected by immunohistochemistry.8.Statistical analysis:all statistical analyses were performed with GraphPad Prism 6 software.Each result was shown as the mean±SD of three independent experiments（n=3）.The differences between the two groups were compared by student t test（Student’ s t test）analysis.The statistical significance of the multi-group comparison was evaluated by one-way variance（ANOVA）analysis and then by Bonferroni analysis.Correlation between the two groups was assessed by Pearson correlation analysis.P<0.05 was considered statistically significant.Results1.The expression levels of TK1 mRNA and protein in T24 cell line were significantly higher than those in 5637 cells（P<0.001）.RT-PCR and Western blot showed that overexpression of TK1 could increase the expression level of TK1 mRNA and protein,while knockdown of TK1 could significantly reduce the expression level of TK1 mRNA and protein.2.TK1 overexpression could increase the expression levels of TFDP1 andβ-catenin mRNA and protein,while TK1 knockdown could inhibit the expression levels of TFDP1,β-catenin mRNA and protein（P<0.001）.Further rescue assays verified that TK1 regulated the expression of β-catenin through TFDP1,and then participated in the regulation of the Wnt/β-catenin pathway.3.The results of CO-IP showed that TK1 and TFDP1 could be enriched at the same time by using anti-IgG antibody and anti-TKl antibody.Meanwhile,we used anti-IgG antibody and anti-TFDP1 antibody for reverse enrichment verification experiment,which could enrich TFDP1 and TK1 at the same time.4.Overexpression of TK1 significantly promoted the proliferation of bladder cancer cells,while TK1 knockdown significantly decreased the proliferation activity of bladder cancer cells（P<0.001）.TK1 overexpression or knockdown could lead to change in the number of cells in S phase and G2/M phase:the number of S phase decreased and G2/M phase cells increased after TK1 was overexpressed in bladder cancer cells,while the number of S phase increased and G2/M phase decreased after TK1 was knocked down in bladder cancer cells.However,TK1 overexpression or knockdown had no significant effect on the apoptosis,migration and invasion ability of bladder cancer cells.5.The proliferation of TK1 overexpressed bladder cancer cells could significantly be inhibited after TFDP1 was knocked down.Meanwhile,the number of S phase increased and G2/M phase cells decreased in bladder cancer cells.However,knocking down TFDP1 had no significant effect on the apoptosis,migration and invasion ability of bladder cancer cells.6.Tumorigenesis in nude mice suggested that TK1 overexpression could significantly promote the proliferation of bladder cancer cells,while TK1 knockdown could significantly inhibit the proliferation of bladder cancer cells.Tumor immunohistochemistry suggested that TK1 overexpression could significantly promote the expression levels of TFDP1 and β-catenin in bladder cancer cells,while TK1 knockdown could significantly inhibit the expression level of TFDP1 and β-catenin in bladder cancer cells.Conclusions1.TK1 could promote the proliferation of bladder cancer cells by regulating cell cycle.2.TK1 could regulate the expression of TFDP1 and β-catenin protein.3.TK1 could regulate Wnt/β-catenin signaling pathway through TFDP1 to promote the proliferation of bladder cancer cellsSignificanceThis study had proved that TK1 was highly expressed in bladder cancer patients by clinical research,and had certain diagnostic value for bladder cancer.TK1 might be a potential marker for proliferation of bladder cancer cells and the expression of TK1 in bladder cancer had an important clinical significance.In order to further verify the relationship between TK1 and cancer,especially with bladder cancer,a multi-dimensional pan-cancer analysis of TK1 single gene had been conducted,which confirmed that TK1 was a good marker for pan-cancer and bladder cancer.Using TCGA,GEO,and Array Express database to analyze the differentially expressed genes in bladder cancer,it was found that TK1 was highly expressed in bladder cancer,and there was a correlation between TK1 and TFDP1.The interaction relationship between TK1 and TFDP1 was inferred and the evidence that TK1 and TFDP1 were associated with cell proliferation was explored by gene co-expression analysis and protein interaction network construction.Furthermore,the interaction relationship between TK1 and TFDP1 was clarified by molecular biology experiment,immune-coprecipitation experiment,cell function experiment and animal experiment.This study investigated the relationship between TK1 and TFDP1 and the phenotype of bladder cancer cells,and discussed the regulatory relationship between TK1,TFDP1 and Wnt/β-catenin.It was concluded that TK1 could promote the proliferation of bladder cancer cells by regulating the cell cycle,the interaction of TK1 and TFDP1 protein affected the downstream Wnt/β-catenin signaling pathway and promoted the proliferation of bladder cancer cells.The significance of this study was to clarify the pathway mechanism that TK1 participated in cell cycle regulation,promoted the proliferation of bladder cancer cells,and provided molecular markers and potential therapeutic targets for clinical diagnosis and treatment of bladder cancer. |
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