The Research On The Role Of B7-H3 In Promotion Of Recurrence And Metastasis In Bladder Cancer Through Regulating Self-renewal Of Bladder Cancer Stem Cells

Background: Bladder cancer is one of the most common malignant tumors of the urinary system that causes male death.Most patients have local recurrence and distant metastasis within 2 years after transurethral resection.At present,the diagnosis and treatment for bladder cancer are limited,and recurrence is still the main reason for the poor prognosis of bladder cancer patients.Cancer stem cell theory is considered to be the root cause of tumor growth,recurrence,metastasis and drug resistance.The tumorigenicity of bladder cancer stem cells is significantly related to the prognosis of bladder cancer.The target therapy of bladder cancer stem cells can provide a new way of treatment for the patients with bladder cancer,and it is very likely to be the key to cure bladder cancer.B7-H3 is a newly discovered T cell costimulatory molecule,and plays a role in the process of tumor immunity,its function in the process of tumor immunity remains controversial.B7-H3 also acts as a new tumor associated antigen which has been confirmed abnormal expression in many malignant epithelial tumors,such as prostate cancer,non-small cell lung cancer,gastric cancer,ovarian cancer,etc.Previous studies have confirmed that the expression level of B7-H3 in tumor tissues is related to clinicopathological grading and prognosis,and is involved in regulating tumor biological function,and is associated with tumor recurrence and metastasis.We aimed to compare with bladder cancer primary tumors and recurrence foci of B7-H3 expression to reveal the correlation between B7-H3 and recurrence and metastasis of bladder cancer,and apply molecular biology method further invesigate whether B7-H3 could affect bladder cancer recurrence and metastasis by regulating bladder cancer stem cell function,and could it be used as therapeutic targets for further study of cancer stem cells and prevention of bladder cancer recurrence and metastasis.Objective: To study the relationship between B7-H3 and recurrence and metastasis in bladder cancer,and evauate the effect of B7-H3 on stem cells of bladder cancer.Method: 1.We collected 156 cases of bladder cancer tissue specimens,including 72 cases of recurrence or metastasis,of which both primary and recurrence tissue specimens had been collected.Half of each tissue was made into wax blocks and the other half was lysed.Immunohistochemistry and enzyme linked Immunosorbent assay(ELISA)were used to detect the expression of B7-H3 in the recurrent and non-recurrent group of bladder cancer to find the correlation between the expression of B7-H3 and clinical prognosis.2.Enrichment of stem cell group(CD20-CD44+ cells)were separated from T24,BIU-87 and 5637 bladder cancer cell lines by immunomagnetic separation using CD44 and CD20 antibody.RT-PCR verified stem cell markers expression of CD44,CD20,CD133,OCT4 and Bmi-1.Model of transplanted tumor in mice was used to verify the strong tumorigenicity of stem cells by serial dilution.We collected three stem cells and three non stem cells for gene sequencing,and RT-PCR verified the test results.3.We used B7-H3 silencing lentivirus and negative control to transfect the T24 stem cell side population of human bladder cancer cells.Then we screened cells to obtain CD20-CD44+ T24 stem cells with B7-H3 stable low expression(B7-H3 KD CSCs)and control group T24 CD20-CD44+ cells(NC CSCs)through puromycin.RT-PCR,western blot and cell fluorescence were used to verify the transfection efficiency.In suspension culture,tumor microsphere formation experiments and nude mice bearing tumor experiments were carried out to observe the effects of B7-H3 on self-renewal,tumorigenicity and invasion and metastasis potential of bladder cancer stem cells.4.Tumor tissues from tumor bearing mice were collected and primary cells were extracted.Cell function tests were carried out to study whether the biological characteristics of primary tumor cells formed by B7-H3 silencing stem cells are consistent with the changes of stem cells,including cell proliferation,invasion,metastasis and drug resistance.Results: 1.The positive rate of B7-H3 in bladder cancer recurrence group was 79.2%,which was significantly higher than that of non-recurrence group(53.6%),P<0.05.ELISA results show that the expression level of secondary foci(158.26±8.13 ng/ml)was significantly higher than that of primary tumor(67.45±5.68 ng/ml),P<0.05.The expression of B7-H3 is related with the the expression of CD44 among the bladder cancer recurrence group,P<0.05.2.The CD20-CD44+ side population cells obtained by the separation of immunomagnetic beads proved to be rich of the stem cells in bladder cancer cell line,which have the ability of self-renewal and strong tumorigenicity.The results of microarray and RT-PCR validation showed that B7-H3 was highly expressed in the stem cell side population compared with the non stem cell side group in the three groups of bladder cancer stem cell lines(P<0.05).3.RT-PCR,WB and cell fluorescence co-verified the efficiency of lentivirus transfection,and the expression of level of B7-H3 in B7-H3 KD CSCs decreased by 4 times compared with that in NC CSCs,and the difference was statistically significant(P<0.05).The experimental results show that compared with the control group,the ability of self-renewal is weaker in B7-H3 silencing bladder cancer stem cells as the number of microspheres decreased,the microsphere diameter became shorter,and one single cell took longer time to form the tumor spheres(P<0.05).Stem cell marker m RNA and protein expression was significantly decreased,and the difference was statistically significant(P<0.05).The experimental results in vivo alao showed that compared with the control group,in B7-H3 silencing stem cells,tumor formation is relatively small,growth ability is weak,the liver metastasis occurred in fewer lesions,even lung without metastasis,with statistically significant difference(P<0.05).4.Transwell experiments showed that the primary tumor cells obtained from xenografts in B7-H3 KD CSCs group had weaker invasion and metastasis ability than those obtained from NC CSCs group(P<0.05).After treated with THP for 24 hours,primary cells of B7-H3 KD CSCs group were more sensitive to THP,with more apoptotic cells detected by flow cytometry.Conclusion:B7-H3 was closely related to the recurrence and prognosis of bladder cancer.Cancer stem cell overexpressed B7-H3 significantly enhanced the self-renewal ability and the malignant biological behavior of tumor cells,promoted the invasion and metastasis of bladder cancer.Targeted intervention of B7-H3 can be used as bladder cancer stem cell therapy and B7-H3 can be used as a specific marker in the early clinical diagnosis to predict the risk of bladder cancer recurrence and provide a new strategy for the treatment of bladder cancer.