The Effect Of Transmembrane Protein 40 On The Malignant Phenotype Of Bladder Cancer Cells And Its Related Mechanism

Bladder cancer is one of the most common malignant tumor in the urinary system.It has the highest morbidity and mortality among the urinary system tumors,which threatens people’s life and health severely all over the world.Although approximately 75%of the patients are group as non-muscle-invasive bladder cancer(NMIBC)with a relatively high 5-year survival rate,other 25%patients belong to muscle-invasive bladder cancer(MIBC)are subjected to subsequent metastatic disease after the first aggressive treatment.According to incomplete statistics,the number of deaths from bladder cancer patients in the world is more than 150 thousand people every year,and the situation is extremely severe.The main clinical treatment of bladder cancer is the use of total bladder surgery,so the recurrence rate is still very high,the effect is not ideal.Therefore,we should understand the molecular mechanism of bladder cancer,perfect the specific diagnosis and treatment methods and find the unique molecular diagnostic markers of bladder cancer,so as to realize the diagnosis and treatment of gene targeting.Then reducing the incidence and mortality of bladder cancer,improving the survival rate and quality of life of patients after 5 years is a difficult problem for today’s medicine.TMEM40 is a transmembrane protein with a size of 34 kDa,consisting of 233 amino acids and contains two domains.In our previous studies,we found that TMEM40 expression was upregulated in bladder tumors,mainly through interaction with downstream related molecules of p53 signaling pathway.At present,the relationship between TMEM40 and bladder cancer is still poorly understood.The role of TMEM40 in prognosis,diagnosis and treatment of bladder cancer still needs further study.First,in the current study we use qRT-PCR and WB to detect the expression of TMEM40 in bladder cancer cells and tissues in mRNA and protein levels.The results showed that TMEM40 expression was upregulated in bladder cancer cells and tissues.Furthermore,through immunohistochemical tissue microarray experiments,to explore the relationship between TMEM40 expression level and bladder cancer patients clinical and pathological parameters.It was found that the expression of TMEM40 was significantly correlated with clinical stage,pT stage and histopathologic grade in 115 patients with bladder cancer,but not correlated with age,sex and so on.Secondly,we selected two human bladder cancer cell lines(5637,EJ)and screened G418 bladder cancer cells with over expression and knockdown by different concentration gradient TMEM40.Finally,a series of functional tests,such as CCK-8,EdU,Transwell,cell cycle,apoptosis and subcutaneous tumor formation in nude mice,were used to confirm the possible influence of TMEM40 bladder cancer phenotype.Cell proliferation experiments show that upregulation of TMEM40 promotes cell proliferation and downregulation of TMEM40 in a certain degree of cell proliferation inhibition;Transwell assay showed that TMEM40 expression promotes cell migration and invasion,while TMEM40 knockdown inhibited cell migration and invasion;flow cytometry the results showed that overexpression of TMEM40 can inhibit the apoptosis of cells,while knockdown of TMEM40 promoted cell apoptosis;overexpression of TMEM40 accelerated phase G1/S,and knockdown of TMEM40 cell cycle arrest in G1 phase.In addition,the results of tumorigenesis in nude mice showed that TMEM40 could significantly inhibit the growth of tumor in nude mice compared with the control group.This study shows that TMEM40 is highly expressed in the tissues and cells of bladder cancer.The expression level is closely related to the malignant phenotype of migration,invasion and proliferation of bladder cancer.Overexpression of TMEM40 makes p53,p21 Cleaved-caspase-3,Cleaved-caspase-9,Cleaved-PARP1 expression down regulated,and the expression of c-myc and CCND1 up-regulated,thus inhibiting 5637 and EJ Cell apoptosis and expedite G1/S transformation.Therefore,TMEM40 plays an important role in the development of bladder cancer,and may be the gene target and molecular marker for the clinical treatment of bladder cancer patients.