| 【Background】Bladder cancer is one of the common malignant tumors with high incidence and poor prognosis.All over the world,there are about 550,000 newly diagnosed patients and approximately 200,000 individuals which is die due to the disease every year.Bladder cancer can be divided into non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC)according to different pathological stages.Transurethral resection of bladder tumor is the main method of treatment for NMIBC.About 60 percent patients experience recurrence in the short term after surgery.Radical cystectomy is the main treatment for MIBC,and the 5-year survival rate of patients is only about 60 percent.When distant metastasis or progression of MIBC is occurred,the 5-year survival rate will be less than 10 percent.There is currently a lack of curative options for patients with advanced and metastatic bladder cancer.In recent years,targeted therapy based on gene expression and sequencing research has played an important role in the treatment of advanced bladder cancer due to its advantages of precise treatment,strong personalization,and low side effects.Therefore,the study of genes and molecular mechanisms closely related to the malignant progression of bladder cancer and the development of new targets for bladder cancer targeted therapy have hit a research hotspot.Protein kinase membrane associated tyrosine/threonine 1(PKMYT1)is a member of the WEE kinase family.It functions as a cell cycle G2/M checkpoint and is involved in DNA damage repair.A large number of studies have confirmed that PKMYT1 is related to tumor biological behaviors such as tumor proliferation,invasion,and radiosensitivity,but there is still a lack of relevant research on the expression and function of PKMYT1 in bladder cancer.【Objective】To explore the expression of PKMYT1,biological function and potential molecular mechanism in bladder cancer.To study its relationship between the clinical prognosis of bladder cancer patients and expression of PKMYT1.【Materials and Methods】1.10 cases of bladder cancer tissues and paired adjacent normal urothelial tissues were undergone transcriptome sequencing,then TCGA and GEO data were analyzed and literatures were searched to screen candidate genes.2.Cancer tissue and adjacent normal urothelial tissue specimens from bladder cancer patients were selected to extract RNA and protein,and to detect the expression of PKMYT1 by western blot and real-time fluorescent quantitative PCR.3.Immunohistochemical staining was performed on 151 clinical patients to detect the expression of PKMYT1,and to explore the relationship between the expression of PKMYT1 and the clinical prognosis of patients.4.The relationship between expression of PKMYT1 and clinical prognosis of patients was externally verified through GEO database.5.The expression of PKMYT1 in four different bladder cancer cell lines and normal urothelial cell lines was detected by real-time quantitative PCR and western blot.The expression of PKMYT1 in J82 cells and UMUC3 cells was knocked down by RNA interference technology,and the proliferation of J82 and UMUC3 cells was detected by clone formation and CCK-8 cell proliferation assays.6.The expression of PKMYT1 was knockdown to explore invasion and migration of bladder cancer cells,J82 and UMUC3,by wound healing and Transwell migration and invasion experiments.7.The effects of PKMYT1 on the apoptosis and cell cycle of bladder cancer cells,J82 and UMUC3,are detected by flow cytometry.8.The differentially expressed genes between the PKMYT1 knockdown group and the control group in J82 cells were analyzed by expression profiling chip sequencing,then it aimed to explore the signaling pathway by which PKMYT1 regulate bladder cancer progression.【Results】1.Transcriptome sequencing of 10 cases of bladder cancer and adjacent normal urothelial tissue shows that the expression of PKMYT1 in bladder cancer tissue is significantly higher than that in paired normal urothelial tissue,and the log2-fold change of differential expression was 1.96.In the sequencing samples,there are 9patients that the expression of PKMYT1 is higher than that in adjacent normal urothelial tissues(P<0.05).In the public database TCGA and GSE45184 data sets,the expression level of PKMYT1 in bladder cancer tissues is higher than that in normal urothelial tissues,and the log2-fold change of its differential expression is greater than 1.5 times.Through literature searching,there is no study on the expression and function of PKMYT1 in bladder cancer.2.10 cases of bladder cancer and paired adjacent normal urothelial tissues are selected to detect the expression of PKMYT1 protein by western blot.The expression of PKMYT1 in 10 cases of bladder cancer tissues is higher than that of matched normal urothelium.The expression of PKMYT1 is detected by real-time fluorescence quantitative PCR in 40 cases,and 32 cases of bladder cancer tissue has higher PKMYT1 expression than matched normal urothelium(P<0.01).3.151 patients performed by immunohistochemical staining,the median survival time of PKMYT1 high expression group is 43.1 months,and that of low expression group is 69.6 months.The overall survival time and cancer-specific survival time of patients with high expression are significantly lower than those of patients with low expression(P<0.001).Univariate COX regression analysis shows that the age(P=0.034),T stage(P=0.001),pathological grade(P=0.017),tumor number(P=0.009),and PKMYT1 expression(P=0.001)of the patients are bladder cancer prognostic risk factors.Multivariate COX regression analysis find that T stage(P<0.001)and high expression of PKMYT1(P<0.001)are independent risk factors for the prognosis of patients.4.GEO data analysis confirms that the overall survival time of patients with high PKMYT1 expression is significantly lower than that of patients with low PKMYT1expression(P<0.05).5.The CCK-8 cell proliferation experiment shows that compared with the negative control group,the cell proliferation in the J82 cell knockdown group is significantly inhibited(P<0.05),while the UMUC3 cell knockdown group is significantly inhibited(P<0.05).In the clone formation experiment,the number of clones formed in the PKMYT1 knockdown group is significantly lower than that in the control group(P<0.05).6.In the wound healing test,the migration ability of J82 and UMUC3 cells in the PKMYT1 knockdown group is significantly lower than that in the control group(P<0.001).In Transwell assay,the migration and invasion ability of J82 and UMUC3 cells in PKMYT1 knockdown group is significantly inhibited(P<0.001).7.Flow cytometry shows that compared with the negative control group,PKMYT1 knockdown groups promote cell apoptosis(P<0.001).Knockdown of PKMYT1,in J82 and UMUC3 cells,decrease the proportion of cells in G2/M phase(P<0.05).8.The microarray analysis of expression profile shows that the differential genes caused by knockdown of PKMYT1 are enriched in PI3K-AKT signaling pathway,TNF signaling pathway,MAPK signaling pathway and other related signaling pathways of tumor occurrence and development.Western blot confirms that knockdown of PKMYT1 reduces the protein expression of phosphorylated ERK and phosphorylated MEK,the molecular markers of MAPK signaling pathway,and inhibits the activation of MAPK pathway.【Conclusion】PKMYT1 is highly expressed in bladder cancer.High expression of PKMYT1 is associated with poor prognosis of patients,and high expression of PKMMYT1 can be used as an independent risk factor for the prognosis of bladder cancer.Knockdown of PKMYT1 expression can inhibit the proliferation,migration and invasion of bladder cancer cells,and promote the apoptosis of bladder cancer cells.At the same time,PKMYT1 is involved in the regulation of cell cycle,and knockdown of PKMYT1 can relieve its G2/M blockade.PKMYT1 promotes the occurrence and development of bladder cancer through the ERK/MAPK pathway. |
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