Association Of Costimulatory Molecule B7-H3 Expression In Urothelial Carcinoma Of Bladder With Clinical Significance And Mechanism Research

At present,bladder cancer is the most common malignant tumor in the urinary system in China,and urothelial carcinoma is the most common pathological type.The search for new tumor markers to improve the diagnosis of bladder cancer and the accuracy of the prognosis is a problem to be solved.B7-H3 is an important member of the B7 family of co-stimulatory molecules and is a typical type I transmembrane protein molecule belonging to the immunoglobulin superfamily.Since B7-H3 demonstrated tumor immune co-regulatory function,many scholars began to pay attention to the expression and clinical significance of B7-H3 in human tumor tissues.Existing studies have shown that B7-H3 molecules may have important value as a tumor-associated antigen in the diagnosis and prognosis of tumors.Part one Association of B7-H3 expression in urothelial carcinoma of bladder with clinical significanceObjective:To investigate association of B7-H3 expression in urothelial carcinoma of bladder(UCB)with clinicopathological features and its clinical significance in tumor development of patients.Methods:1.The expression levels of B7-H3 in tumor specimens from 134consecutive patients undergoing partial resection or radical cystectomy for UCB were tested using immunohistochemistry.2.The relationships between B7-H3 expression and clinicopathological parameters for UCB were evaluated using Chi-square test and Fisher exact test.3.The associations of B7-H3 expression with patient outcome after partial resection or cystectomy were estimated using the Kaplan-Meier survival analysis.Results:1.In 134 UCB patients,B7-H3 was expressed positively by 93/134patients(69.4%),which was significantly higher than in adjacent nontumor urothelium and normal bladder tissues(15/60 cases,25.0%;11/60 cases,18.3%;P<0.05).Among urothelial carcinoma tissues,B7-H3 expression had no statistcal association with gender,age,tumor grade,tumor stage,recurrence,lymph node metastasis(P>0.05);but the rates of vascular invasion and distant metastasis in patients with high B7-H3 expression were promitently higher than in patients with low B7-H3 expression(69.9%vs.46.3%;49.5%vs.26.8%),indicating that B7-H3 expression was significantly associated with advanced vascular invasion and distant metastasis(P=0.009;P=0.015).2.Kaplan-Meier survival analysis revealed that among the 134 UCB patients,overall survival(OS)and progression-free survival(PFS)were significantly shorter in the high B7-H3 positive expression group compared with the low B7-H3 positive expression group(P=0.002;P=0.020).These differences were even more significant in muscle-invasive UCB patients(P<0.001;P<0.001).Summary:1.Patients with high expression of B7-H3 in bladder cancer tissues had a higher proportion of vascular invasion and distant organ metastasis,and the postoperative survival time was significantly shorter than that in patients with low B7-H3 expression.2.The expression level of B7-H3 molecule can be used as an effective predictor of postoperative tumor progression and poor prognosis in patients of bladder cancer.Part two Correlations of B7-H3 expression and CD163~+TAMs infiltration in urothelial carcinoma of bladder with prognostic significanceObjective:To investigate correlation of B7-H3 expression and CD163~+TAMs infiltration in UCB patients with prognostic significance for predicting the risk of tumor progression and death.Methods:1.CD163~+TAMs infiltration in tumor specimens from 134 consecutive patients undergoing partial resection or radical cystectomy for UCB were tested using immunohistochemistry.2.The association between CD163~+TAMs infiltration with B7-H3expression and clinicopathological features for UCB were evaluated using Spearman rank correlationChi-square test and Fisher exact test as apropriate.3.Kaplan-Meier method and Cox proportional hazards regression analysis were used to explore the independent or synergistic effects of B7-H3expression and CD163~+TAMs infiltration on tumor progression and death risk.Results:1.In 134 UCB patients,CD163~+TAMs infiltration had no statistcal association with gender,age,tumor grade,tumor stage,recurrence,lymph node metastasis(P>0.05);but the rates of vascular invasion and distant metastasis in patients with high CD163~+TAMs infiltration were promitently higher than in patients with low CD163~+TAMs infiltration(74.6%vs.53.3%;52.5%vs.34.7%),indicating that CD163~+TAMs infiltration was significantly associated with advanced vascular invasion and distant metastasis(P=0.012;P=0.038).2.Spearman rank correlation analysis showed that there was positive association between B7-H3 expression and CD163~+TAMs infiltration among the 134 patients with bladder urothelial carcinoma(r=0.230,P=0.007).Kaplan-Meier survival analysis revealed that overall survival(OS)and progression-free survival(PFS)were not statistically correlated to CD163~+TAMs infiltration(P=0.074;P=0.090).The results from Cox proportional hazards regression analysis for the 134 UCB patients revealed that there were no any significant associations between CD163~+TAMs infiltration with the risk of overall mortality,tumor-specific mortality and tumor progression(P=0.290;P=0.525;P=0.355).Interestingly,in subgroup of patients with high CD163~+TAMs infiltration,high B7-H3 expression was associated with worse outcome such as overall mortality,tumor-specific mortality and tumor progression than low B7-H3 expression(P=0.002;P=0.004;P=0.018),while this association was not observed in subgroup of patients with low CD163~+TAMs infiltration.The overall mortality,tumor-specific mortality and risk of tumor progression were significantly elevated in the high B7-H3 expression group among the 134 patients(P=0.001;P<0.001;P<0.001).But significant results were not observed in the subgroup of 55 localized bladder cancers(P=0.138;P=0.269;P=0.147).Summary:1.In bladder urothelial carcinoma,patients with high infiltration of CD163~+TAMs have a higher proportion of vascular invasion and distant organ metastases.2.There was significantly positive correlation between CD163~+TAMs infiltrations and B7-H3 expression.In the subgroup of high CD163~+TAMs infiltration,the overall risk of death,the risk of tumor-specific death,and the risk of tumor progression in patients with high B7-H3 expression were significantly higher than those with low B7-H3 expression.3.The degree of CD163~+TAMs infiltration can be combined with the expression level of B7-H3 as a predictor of mortality risk after partial or radical resection of bladder cancer.Part three Mechanism research on the malignant behavior of T24 cell with B7-H3 knockdown by RNA interferenceObjective:To investigate the changes of proliferation and invasion capacity after B7-H3 silencing of T24 cell and explore the probable influence mechanism.Methods:1.B7-H3 silencing of the T24 cell line was performed using small interfering(si)RNA recombinant lentiviral vector as LV-B7-H3-RNAi.2.Western blot and RT-PCR assay was performed to evaluate the expression of B7-H3,MMP-2 mRNA and protein in T24 cells transfected by B7-H3 siRNA.3.The proliferation capacity of T24 cell line was evaluated by MTS assay.4.The invasion capacity of T24 cell line was evaluated by transwell assay.Results:1.The recombinant lentiviral vector carrying small interfering RNA(siRNA)was stably transfected into T24 cells of bladder urothelial carcinoma.T24/B7-H3 siRNA cell line with low B7-H3 expression was successfully established.2.B7-H3 mRNA expression was significantly decreased in the T24/B7-H3 siRNA group(0.0952±0.0012)compared with T24/control siRNA group(0.4638±0.0010)and untransfected T24 control group(0.4650±0.0008)and the differences were statistically significant(P<0.01;P<0.01).3.B7-H3 protein expression was significantly decreased in the T24/B7-H3 siRNA group(0.1364±0.0015)compared with T24/control siRNA group(0.1772±0.0008)and untransfected T24 control group(0.1783±0.0019)and the differences were statistically significant(P<0.01;P<0.01).4.MMP-2 mRNA expression was significantly decreased in the T24/B7-H3 siRNA group(0.0654±0.0031)compared with T24/control siRNA group(0.4340±0.0028)and untransfected T24 control group(0.4360±0.0005)and the differences were statistically significant(P<0.01;P<0.01).5.MMP-2 mRNA expression was significantly decreased in the T24/B7-H3 siRNA group(6.0190±0.1339)compared with T24/control siRNA group(16.0327±0.0952)anduntransfectedT24controlgroup(16.1658±0.2902)and the differences were statistically significant(P<0.01;P<0.01).6.MTS assay showed that the cells proliferation was significantly decreased in the T24/B7-H3 siRNA group compared with T24/control siRNA group and untransfected T24 control group and the differences were statistically significant(P<0.01;P<0.01).7.Transwell assay showed that the cells invasion capacity was significantly decreased in the T24/B7-H3 siRNA group compared with T24/control siRNA group and untransfected T24 control group and the differences were statistically significant(P<0.01;P<0.01).Summary:1.In this part of the experiment,T24/B7-H3-siRNA cell line with low B7-H3 expression was successfully established.2.The cell proliferation and invasion ability of T24/B7-H3-siRNA group were significantly inhibited,suggesting that B7-H3 is involved in the progression and metastasis of bladder cancer.Conclusions:1.B7-H3 highly expressed in urothelial bladder cancer compared with adjacent nontumor urothelium and normal bladder tissues.The level of B7-H3expression could be used as the predictor of tumor progression and poor prognosis in UCB patients undegoing partial resection or radical cystectomy.2.B7-H3 expression was positively associated with the dentisy of CD163~+TAMs infiltration.Both B7-H3 and CD163~+TAMs might be conjointly involved in the tumor initiation and progression of UCB.3.Downregulation of B7-H3 by RNA interference significantly decreased T24 cell proliferation and invasion capacity.B7-H3 may participate in the malignant progressive behavior of T24 cell.These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in urothelial bladder cancer.