Effect Of DC/IL-23 On Biological Behavior Of Bladder Cancer And Its Mechanism

1.BackgroundIn China,bladder cancer is a urinary tract tumor disease with the highest incidence.About 150 years ago,Virchow proposed the hypothesis that inflammation is a predisposing factor for tumorigenesis.This point of view originated from his observation that cancerous tissues often caused localized chronic inflammatory reactions,and that inflammatory cells were often present in the removed tumor tissues.In 1970,Burnet proposed the theory of immune surveillance: the immune system can spontaneously identify and remove tumor cells,protect the body tissues,and can play the role of anti-tumor development.The epidemiological survey results in the past 10 years show that patients who are susceptible to chronic inflammatory disease have a higher risk of cancer;about 15% to 20% of cancer deaths are associated with infection and inflammation worldwide.This survey result supports Virchow's view to some extent.Although there are reports about the role of the immune system in inhibiting or promoting cancer,a large amount of data suggests that the immune response in tumor microenvironments is more likely to promote tumor growth,and the immunosuppressive effect induced by tumor microenvironment is much greater than the effective anti-tumor effect by the immune system.The molecular mechanism of tumor-related inflammation is gradually revealed,but it is not fully clear,and the identification of target molecules is the key to improve the diagnosis and treatment of tumors.The fact that different subpopulations of immune effector cells and immunoregulator y cells exist in the tumor microenvironment demonstrates the complexity of the tumor immune environment.Tumor-induced immune cell differentiation and functional changes make this unique environment more complex and varied.Dendritic cells?DCs?are a grou p of special cells,which are bridges between the innate immune response and the adaptive immune response: capturing foreign antigen,migrating to the lymphatic organs,and presenting the antigen to the adaptive immune cells.DCs are currently the most eff ective antigen presenting cells to induce the initial immune response and are heterogeneous cell populations.They belong to a subpopulation with different phenotypes and functions,and play the role of tumor promotion or tumor suppression in a particular environment.DCs play a key role in the initiation and maintenance of antitumor immunity,but in the tumor microenvironment,the strong antigen presentation function efficiency of DCs is significantly reduced or failed.There are many studies about the function and low efficiency of DCs in the tumor microenvironment,and the possibility of various signaling pathways and related abnormal factors related to functional abnormalities are proposed,however,there is no feasible therapeutic method for preventing or reversing the DC dysfunction of tumor patients.Further studying the DC immunology in tumor environments is critical to improve the relative treatment of tumors invasive DC function.IL-23 is an important proinflammatory cytokine,mainly secreted by DCs.So far,the role of IL-23 in tumor microenvironment remains controversial.IL-23 is a STAT3-regulated gene and also a STAT3 agonist.It is noteworthy that the sustained activation of S TAT3 in tumor cells can be spreaded to the surrounding environment,and activate a series of tumor-promoting reactions.Cell stress or cell death can induce the release of inflammatory mediators such as IL-6,ATP,prostaglandin?PG?E₂,heat shock proteins?HSPs?into the tumor microenvironment,and promote DC to secrete IL-23.Interestingly,PGE₂ can down-regulate LPS-stimulated monocytes to secrete IL-12.ATP activates the P2 X purinoreceptor 7?P2RX7?receptors in DCs,resulting in activation of NOD-like receptor pyrin domain-containing 3?NLRP3?and secretion of IL-1?.Thus,it is likely that DC or macrophages secrete IL-23,IL-6 and NLRP3-associated IL-1?,promote Th17 cell differentiation,and induce IL-17-secreting immune response rather than promoting anti-tumor Th1 type immune response.Therefore,the activation of NLRP3 induced by dangerous signals and the role of IL-23 in tumor immunity are worthy to be further studied.Therefore,in this study,we investigate the expression and distribution of DC / IL-23 in human bladder urothelial carcinoma and the biological mechanism of IL-23 on bladder urothelial carcinoma cells..2.Distribution feature of DC/IL-23 in human bladder urothelial tumors2.1 ObjectiveTo study the distribution of DC subpopulation and IL-23 in human bladder urothelial carcinoma.2.2 MethodsThe immunohistochemistry and real-time PCR were used to detect the distribution and expression of IL-23,IL-6 and IL-17 in human bladder urothelial carcinoma.And the expression feature of IL-23 A in bladder tumor tissues and its clinical significance were further confirmed by bioinformatics analysis.2.3 Results2.3.1 Only CD1a⁺,CD11c⁺,CD209⁺DCs subgroups were found in the control group,while CD123⁺ p DCs and CD83⁺ mature DCs sub-population were almostly completely deficient.2.3.2 The number of CD11c⁺,IL-23p19⁺m DCs,CD123⁺p DCs,CD1a⁺,CD209⁺ immature DCs and CD83⁺ mature DCs in bladder tumor tissues were significantly increased.2.3.3 The number of CD11c⁺,CD123⁺,CD1a⁺,CD209⁺DCs in muscle invasive bladder tumor tissues was significantly higher than that in non-muscule invasive bladder tumor group,but the number of CD83⁺ DCs showed a decreasing trend.2.3.4The expression of IL-23,IL-23 R,IL-17 and IL-6 in human bladder urothelial cancerous tissues was significantly higher than that in the adjacent noncancerous tissues.2.3.5 Compared with normal bladder tissues,the expression level of IL-23 A in nonmuscle invasive urothelial cancerous tissues was not significantly increased,but the expression level of IL-23 A in cancer tissues and muscle invasive urothelial cancerous tissues was significantly increased.2.3.6 Compared with the corresponding adjacent tissues,the expression level of IL-23 A in non-muscle invasive urothelial cancerous tissues was significantly increased,which has no significant difference when compared with the muscle invasive urothelial carcinoma group.2.3.7 Compared with Grade2,the IL-23 A expression in Grade 3 bladder urothelial cancerous tissues was significantly increased.2.3.8 Kaplan-Meier analysis suggested that the expression of IL-23 A in clinical tissue samples was negatively correlated with tumor stage and patient prognosis.2.4 Conclusion2.4.1 Under physiological state,DC existed in the organization mainly in the immature state and maintained its immune tolerance;under tumor microenvironment,a large number of immune cells and inflammatory cell were infiltrated,but had dysfunction;higher malignancy degree could decrease the number of CD83⁺ mature DC activated by tumor-specific immune response,which was associated with DC disability and tumor cell immune escape mechanism under the tumor microenvironment.2.4.2 The expression of IL-23,IL-23 R,IL-17 and IL-6 were significantly increased in tumor tissues.The expression of IL-23 A was positively correlated with tumor grade and staging,and negatively correlated with patients' prognosis.With the increase of malignancy,the expression level of IL-23 in adjacent tissues was increased,which was significantly higher than that in normal bladder tissues.This may be an important factor to promote the progression and recurrence of bladder cancer.Therefore,the expression of DC subgroups and IL-23 in bladder tumor tissues was significantly correlated with tumor grade and staging,and was negatively correlated with patients' prognosis,which may be related to the high recurrence and immune escape mechanism of bladder tumor.3.Effect of different concentrations of IL-23 on the biological behavior of bladder urothelial carcinoma cells3.1.ObjectiveTo investigate the biological effect of IL-23 on bladder urothelial carcinoma cells.3.2 MethodsThe effects of different concentrations of IL-23 on proliferation,migration and invasion of bladder tumor cells were observed.3.3 Results3.3.1 Low concentration of IL-23?20ng/ml?promoted the proliferation,migration and invasion of T24 urothelial carcinoma cells and up-regulated the expression of Ki67 and PCNA,while high concentration of IL-23?40ng/ml?played an inhibitory effect.3.3.2 Low concentration of IL-23?20ng/ml?induced HUVEC angiogenesis,while high concentration of IL-23?40ng/ml?played an inhibitory effect.3.4.Conclusion3.4.1 IL-23 regulated T24 cell proliferation and EMT changes in a concentration-dependent manner.3.4.2 IL-23 modulated HUVEC angiogenesis in a concentration-dependent manner.4.Mechanism of IL-23 in regulating EMT transformation of T24 cells4.1 ObjectiveTo study the role of mi R-200b-3p in IL-23 in regulating EMT transformation of T24 cells.4.2 Methods4.2.1 The proliferation of T24 cells and the change of EMT-related proteins were observed under the regulation of IL-23.4.2.2 According to the literature reports and real-time PCR verification,mi RNA with significant correlation were selected,and the biological role of different concentrations of IL-23 on the T24 cells with overexpression of mi RNA was observed.4.3 Results4.3.1 IL-23 with concentration of 20ng/ml up-regulated the expression of T24 cell mesenchymal markers N-cadherin,OB-cadherin and Vimentin and down-regulated the protein expression of E-cadherin in epithelial cells;IL-23 40 ng / ml played an opposite role.4.3.2 Overexpression of mi R-200-3p can inhibit the proliferation and migration of T24 cells induced by 20 ng / ml IL-23 and inhibited the up-regulated expression of N-cadherin and Vimentin induced by IL-23.Reversing the IL-23-induced E-cadherin expression down-regulated by 20ng/ml IL-23.4.4 ConclusionIL-23 influences the mi RNA200b-3p and regulates the proliferation,migration and EMT phenotype of T24 cells in a concentration-dependent manner.5.Summary5.1 DCs exist in non-tumor tissues mainly in CD1a⁺,CD11c⁺,CD209⁺DCs subsets;under tumor microenvironment,the number of CD11c⁺IL-23p19⁺m DCs,CD123⁺ p DCs,CD1a⁺,CD209⁺immature DCs and CD83⁺ mature DCs were significantly increased;higher malignancy is associated with the decreased number of CD83⁺ mature DCs.The possible mechanisms are as follows: DC is mainly immature in tissues and maintains autoimmune tolerance in the physiological state;a large number of immune cells and inflammatory cells are infiltrated in the tumor microenvironment,but have dysfunction;the higher the degree of malignancy,the less the number of CD83⁺ mature DCs that activate tumor-specific immune responses would be,which is associated with DC disability in tumor microenvironment and immune escape mechanism of tumor cells.5.2 The expression of IL-23,IL-23 R,IL-17 and IL-6 is significantly increased in tumor tissues,and the expression of IL-23 A is positively correlated with the grade and staging of the tumor and is negatively correlated with patients' prognosis.The expression level of IL-23 A in tumor-adjacent tissues is significantly higher than that in normal bladder tissues,which might be an important factor to promote the progression and recurrence of bladder cancer.5.3 IL-23 regulates the proliferation,migration and EMT phenotype of T24 cells in a concentration-dependent manner,and this regulation is affected by the expression level of mi RNA200b-3p.