The Study Of Role And Mechanism Of Yes-associated Protein 1 In The Development Of Bladder Cancer

Objective Bladder tumor is the most common type of tumor in the urinary system that has a high recurrence rate,and most of the patients die of bladder cancer metastasis and varying degrees of recurrence.The research of this tumor has aroused widespread concern both at home and abroad.Yes-associated protein 1(YAP1),as an oncogene,plays an important role in the inhibition of apoptosis,loss of cell contact inhibition,promotion of abnormal cell proliferation and malignant transformation of cells.At present,YAP1 is found to be highly expressed in various tumor tissues and is a potential target for early diagnosis of tumor and a potential target for tumor therapy.However,there are few reports on the role of YAP 1 in bladder cancer cells.Therefore,this study aims to observe the role of YAP 1 in the development of bladder cancer and to explore its mechanism of action.Methods The eukaryotic transcription plasmid pGreenPuro was used to establish a recombinant transfection plasmid targeting YAP1 gene.Four kinds of YAP1 shRNA plasmids with different sequences and YAP1 shRNA NC containing no YAP1 sequence were transfected into T24 cells respectively.48h later,the YAP1 expression levels were detected by qPCR and the shRNA plasmids with the best YAP1 silencing effect were screened for T24 cells.The effects of shRNA plasmids on the proliferation of T24 cells were detected by MTT assay.The scratch test was used to detect the effect on the migration ability and the invasion ability was detected by Transwell chamber in vitro.18 nude mice were used to construct human bladder cancer nude mice model,and then divided into 3 groups:YAP1 shRNA3-pGreenPuro group,Control-shRNA group and saline group,with 6 rats in each group.Rats in YAP1 shRNA3-pGreenPuro group were injected with YAP1 shRNA3-pGreenPuro recombinant plasmids,each containing 50μg plasmid+ 400μl 5%agarose+10μl transfection reagent(transfection reagent preparation in instructions).Rats in the Control shRNA group and Saline group were injected with 50μg of negative control plasmid and 50μl of sterilized saline respectively.The tumor histopathology of each group was observed by light microscopy and the expression of YAP1 mRNA in each group was detected by RT-PCR.The protein expression of tumor tissue YAP1 and downstream target genes(IGF,Akt,Jag-1,TGF-β)in each group was detected by western blot.52 cases of bladder cancer and para-cancerous tissues were collected from Department of Urology in our hospital from February 2015 to October 2017,40 samples of bladder inflammation were meanwhile collected.The expression of YAP 1 protein in bladder cancer tissues,adjacent tissues and cystitis tissues was detected by immunohistochemistry and then compared.The correlations between the expression level and clinicopathological features such as age,gender,histological grade,tumor number,tumor diameter,lymph node metastasis,clinical stage and so on were also analyzed during this process.Results 1.YAP1 gene was successfully detected by qPCR in T24 cells and different YAP1 shRNA plasmids were successfully transfected into T24 cells by Lipofectamine 2000.qPCR detection results indicated that YAP1 shRNA3 plasmid had the most significant expression in silencing the YAP1 expression(the inhibition rate was 75.5%),then T24 cells were interfered by YAP1 shRNA3 plasmid,the results of MTT detection found that the proliferation ability of T24 cells transfected with YAP1 shRNA3 plasmid was lower than that of the blank control group(the proliferation rate of blank control group was 85.9%,p<0.01),the migration ability of T24 cells transfected with YAP1 shRNA3 plasmid was weaker than that of the blank control group(widths of 24h scratch were 220μm and 80μm,respectively,p<0.01),the invasion ability of T24 cells transfected with YAP1 shRNA3 plasmid was weaker than that of the blank control group(the average number of cells penetrating the Transwell chamber was 62:345,p<0.01);2.Rats in YAP1 shRNA3-pGreenPuro group,Control-shRNA group and normal saline group all survived during the treatment,and all had tumor formation.The daily diet of nude mice in each group showed no significant changes,the mental state was good,and the body weight did not reduce significantly.The nude mice in each group showed no significant difference in blood routine,liver and kidney function,and no significant abnormality in heart,liver,kidney,spleen and other vital organs was observed in nude mice in each group;3.The tumor size of rats of YAP1 shRNA3-pGreenPuro group[(202.7±37.5)mm3]was significantly smaller than that of Control-shRNA group(792.4±46.7mm3)and saline group(798.6 ± 50.2 mm3)(P<0.05).The tumor inhibition rates of YAP1 shRNA3-pGreenPuro group,Control-shRNA group and saline group were 74.2%,3.7%and 3.6%respectively;4.Microscope observation found that the tumor cells in Control-shRNA group and saline group grew well,the morphology of tumor cells showed irregular expression,and there were more tumor cell mitosis,while tumor growth in YAP1 shRNA3-pGreenPuro group had obvious inhibition,the mitosis of tumor cells was significantly reduced when compared with that in Control-shRNA group and saline group.Tumor necrosis area was found in the YAP 1 shRNA3-pGreenPuro group,in which the cell structure disappeared,with cell disintegration fragments and other performance;5.The expression of YAP 1 mRNA in YAP1 shRNA3-pGreenPuro group was significantly lower than that in Conrtrol-shRNA group and saline group(P<0.05),but there was no significant difference between Control-shRNA group and saline group(P>0.05);6.The expression of tumor cell YAP1 and downstream target protein(IGF,Akt,Jag-1,TGF-β)in shRNA3-pGreenPuro group was significantly lower than that in the Control-shRNA group and saline group(P<0.05),and there was no significant difference between Control-shRNA group and saline group(P>0.05);7.The positive expression rates of YAP1 in bladder cancer group(80.77%,42/52)were significantly higher than those in adjacent non-cancerous bladder cancer group(46.15%,24/52)and bladder inflammation group(20.0%,8/40)(P<0.05),and the positive expression rates of YAP 1 in bladder cancer group(46.15%,24/52)was significantly higher than that in bladder inflammation group(20.0%,8/40)(P<0.05);8.There was no significant correlation between the expression level of YAP1 and the age,sex,histological grade and tumor number(P>0.05),but had significant correlation with tumor diameter,lymph node metastasis and clinical stage(P<0.05).Conclusion YAP1 shRNA3-pGreenPuro can significantly reduce the expression of YAP1 gene in human bladder cancer T24 cells and inhibit the proliferation,migration and invasion of tumor cells,by reducing the expression of YAP1 and downstream target genes(IGF,Akt,Jag-1,TGF-β)it could significantly inhibit the growth of human bladder cancer in nude mice.In addition,the expression level of YAP1 in bladder cancer patients was significantly correlated with tumor diameter,lymph node metastasis and clinical stage(P<0.05),which could serve as a new target in predicting the condition and treatment of bladder cancer.