Expression Of BUB1 In Bladder Cancer And Its Significance And The Mechanism Research Of BUB1 Phosphorylating STAT3

Objective To study the expression of BUB1 in bladder cancer;analyze the correlation among BUB 1,clinical cases of bladder cancer patients and recurrence of bladder cancer.To study the role of BUB1 in regulating the stem cell potential of bladder cancer cells.and analyze the relevance between BUB1 and other important stem cell transcription factors.To investigate the mechanism of BUB1 phosphorylating STAT3.Methods 62 tissue samples of bladder urothelium carcinoma and 40 tissue samples of normal bladder mucosa were collected to identify the expression of BUB1;Western-blot was applied to detect four groups of the bladder cancer tissues and the BUB 1 expression of normal tissues beside the carcinoma from one patient.qRT-PCR was used to detect the tumor tissues and the BUB 1 mRNA expression of normal bladder tissues from 68 patients with bladder cancer.Upon the test results,clinical data and flow-up results,the correlation between BUB 1 expression and clinic pathologic stage and level as well as recurrence possibilities of bladder cancer.We choose CD44 and CD24 as stem cell marker,using FCM to find tumor stem cell in EJ、T24 and HTB-9 cells.We compare the expression of important stem cell transcription factors after we knock down BUB1 through qRT-PCR.Then we conduct Sphere formation assay to find out whether BUB1 has influence on stem sell formation.We also use animal tumor model to verify our findings.Campare the expressions of BUB1 and STAT3 in bladder cancer tissue and normal bladder tissue through Immunofluorescence methods to find the relevance of them.We design different plasmid of STAT3 to find out the connect-point of BUB1 and STAT3 through CoIP.Then we use lusiferase to test the activity of SOX2 promotor region in different BUB1 expression level.We use IHC and western–blot to investigate the protein expression level in animal tumor model.Results Immunohistochemical result indicated that the positive expression of BUB 1in bladder cancer cell was apparently higher than that in normal bladder mucosa cell(p<0.001).Stratified analysis result showed that the positive expression of BUB 1 in MIBC was higher than NMIBC(p<0.05).The result of Western-blot identified that the expression of BUB 1 in the bladder cancer tissue(0.657±0.213)was obviously higher than the expression in the normal tissue beside the carcinoma(0.174±0.182)(t=3.440,p=0.0413)from one patient.Similarly,the result of qRT-PCR demonstrated that the expression of BUB 1 in the bladder cancer in mRNA level was also clearly higher than that in the normal bladder mucosa tissue beside the carcinoma(3.86±0.869 versus 1.38±0.483,p<0.001).Considering the patient’s material,it is analyzed that the expression of BUB 1 in MIBC(T2-T4)was notably higher than that in NMIBC(T1);the expression of BUB 1 has no obvious relation with the gender,age,tumor size and tumor amount of the patient(p>0.05).Through the Kaplan-Meier survival curve,it is found that bladder cancer recurrence rate of the patient with high expressed BUB 1 was significantly higher than the patient with low BUB 1(p=0.0334).We choose CD 44~+/CD24~-phenotype as stem cell potential type and we find 5.02%HTB-9 cells match the phenotype.The outcomes of qRT-PCR show the expression of SOX2、OCT4 and Nanog decreased after knock down BUB1.Sphere formation assay indicate BUB1could increase the number of sphere cell.Immunofluorescence test shows the expression of BUB1and STAT3 has relevance in bladder cancer tissue and normal bladder tissue.And we find that BUB1 can connect STAT3 on the site of 586-770 Amino acid fragment of STAT3.Besides,we find BUB1can phosphorylating STAT3 in the western–blot experiment.The outcome of Luciferase test demonstates BUB1can promote the activity of SOX2 promotor region.Also,the expression of BUB1、STAT3、SOX2、Ki-67 and CD44 decreased in the2OH-BNPP1 group of the animal tumor model.Conclusion The expression of BUB 1 in the bladder cancer tissue is obviously higher than that in the normal bladder mucosa tissue beside the carcinoma,and the expression is related with clinic pathologic stage and level,while the expression has no relation with the gender,age,tumor size and tumor amount of the patient.The patient with high expressed BUB 1 has higher possibility of bladder cancer recurrence.5.02%HTB-9 cells match CD 44~+/CD24~-phenotype which indicate the existence of tumor stem cell in bladder cancer cells.BUB1 could regulate the expression level of SOX2、OCT4 and Nanog.Our finding indicate that BUB1 can connect STAT3 on the site of 586-770 Amino acid fragment of STAT3.BUB1 can phosphorylate STAT3.Actvated STAT3 can promote the activity of SOX2 promotor region.