The Role And Mechanism Of P3H4 Gene In Proliferation And Invasion Of Bladder Urothelial Carcinoma

Background:Bladder urothelial carcinoma(BUC)is the most common malignancy of the genitourinary system.Surgical resection combined with radiotherapy or chemotherapy has been used in the treatment of BUC.However,patients who under went active treatment are still struggling with the current situation of easy recurrence,poor prognosis,low survival rate and poor quality of life.Therefore,it is urgent and important to find new and effective therapeutic targets.Prolyl 3-hydroxylase family member 4(P3H4)was initially identified as a protein related to synaptonema 1 complex(SC),which is closely related to human cancers.However,the relationship between P3H4 gene and BUC remains to be uncertain.Objective:To study the role of P3H4 in bladder cancer and the regulatory mechanism affecting its expression.Methods:Based on the expression data of 408 cases with bladder cancer and 19 cases with normal tissue samples in TCGA database,T-test was used to observe the difference of P3H4 gene expression between cancer and normal tissue samples.According to TCGA bladder cancer sample expression data and clinical information,univariate and multivariate cox regression analysis was used to observe the correlation between P3H4 gene expression and survival time.log-rank test was used to observe the survival difference between low-risk group and high-risk group,and KM survival curve was drawn.The expression pattern of P3H4 in BC tissue was detected by online data and immunohistochemical analysis.Secondly,the effect of low P3H4 expression on the biological function of BC cells were examined by CCK8,colony formation,flow cytometry,Tranwell and scratch method.The nude mouse model with xenotransplantation was established to detect the effect of P3H4 knockdown on BC growth in vivo.In terms of mechanism,the transcription factors binding to P3H4 promoter were predicted through the database.The transcription factors directly binding to P3H4 promoter were identified by qRT-PCR,western blot and dual-luciferase reporter assay,and the binding sites were screened.Finally,rescue experiments were conducted to evaluate the effect of transcription factors regulating P3H4 expression on the biological function of BC cells.Results:Compared with normal samples,P3H4 gene expression was significantly higher in bladder cancer samples.Compared with papillary bladder cancer samples,P3H4 gene expression was significantly higher in non-papillary bladder cancer samples.Compared with low-grade bladder cancer samples,P3H4 gene expression was significantly higher in high-grade(Stage Ⅲ/Ⅳ)samples.Compared with bladder cancer samples without lymph node metastasis,P3H4 gene expression was significantly higher in lymph node metastasis samples.Univariate cox analysis was used to calculate the correlation between P3H4 gene expression and overall survival of bladder cancer patients,and the results showed that P3H4 gene expression was significantly correlated with survival time of bladder cancer patients(P=0.0006).HR>1 indicated that P3H4 was a risk factor,and its high expression was correlated with poor prognosis of patients.Multivariate cox analysis was used to observe the correlat ion between clinical variables and overall survival,and calculate whether P3H4 gene can be used as an independent prognostic factor to predict the survival of bladder cancer patients,and the results showed that the stage stage and P3H4 gene expression were significantly correlated with the survival time of bladder cancer patients(P<0.05).In addition,after correcting for age,gender,stage and other clinical factors,the expression of P3H4 gene was still significantly correlated with the prognos is of patients,indicating that P3H4 can be used as an independent prognostic factor to predict the survival of patients.In the high-risk group with high P3H4 gene expression,the overall survival time was significantly lower than that in the low-risk group(log-rank p=0.0067),indicating that P3H4 gene expression can be used as an index to predict survival.The abnormally high expression of P3H4 mRNA and protein in bladder cancer tissues was confirmed by database analysis and immunohist ochemical results.Further knock-down of P3H4 gene could arrest the cell cycle of EJ and T24 cells in G1 phase,thus inhibiting cell proliferation.However,it had no effect on apoptosis.In addition,knock-down of P3H4 also inhibited migration and invasion of EJ and T24 cells by inhibiting EMT process.The nude mouse model with xenotransplantation also showed that P3H4 knockdown inhibited the growth of subcutaneous bladder tumor,indicating that P3H4 gene may play a proto-oncogene function in bladder progression.By further exploring its molecular mechanism,it was found that transcription factor ETV4 could regulate P3H4 gene transcription in EJ and T24 cells.ETV4 could directly bind to the promoter region of P3H4 gene and regulate its transcriptional activity.Conclusions:This study confirms that P3H4 gene expression is significantly high in bladder cancer samples by bioinformatics analysis,and P3H4 can be used as an independent prognostic factor to predict the survival of patients.The role of P3 H4 gene in bladder cancer is confirmed by in vivo and in vitro experiments,and it is found that P3H4 gene may play a proto-oncogene function in bladder progression.Therefore,as far as its molecular mechanism is concerned,it can be concluded that transcription factor ETV4 can directly bind to the promoter region of P3H4 gene and regulate its transcriptional activity.