Clinical Significance And Functional Mechanism Of IL-33 On Esophageal Squamous Cell Carcinoma

Background and purposeEsophageal cancer is the one of the most common malignancies in the world,ranking 8th in the global incidence of malignant tumors,and the 6th place in mortality,which seriously endangers human health.There are two main types of esophageal histology,esophageal squamous cell carcinomas(esophageal squamous cell carcinoma,ESCC)and esophageal adenocarcinoma(esophageal adenocarcinoma,EAC),which differ remarkeabley on regional distribution and race.In China,most of esophageal carcinomas are ESCC,which accounts for more than 90%of all esophageal cancer.Although great progress has been achieved in recent years for malignant tumor targeting therapy,immune therapy,in addition to surgery,radiation therapy and chemotherapy,for esophageal cancer,there is no evidence-based medicine proved effective targeted or immune therapy.So the study on the molecular mechanism of ESCC occurrence and development becomes extremely important for treatment of ESCC.Interleukin-33(IL-33)is a newly discovered multi-functional cytokine.Because of the similarity of its gene sequence with Interleukin 1 beta(IL-1 beta)and Interleukin-18 sequence,it is classified as members of IL-1 cytokine family.IL-33 can functin as a secretory cytokine in combination with orphan receptor ST2,and can also function to play its role as a transcription factor in the nucleus.At present,the role of IL-33 in tumor development,especially in ESCC,has not been fully elucidated.In tumor microenvironment,chemokine has a small molecular weight,participates in the directional migration of inflammatory cells and immune cells immune regulatory factor,and plays an important role in tumorgenesis.CCL2,also called the MCP-1(monocyte chemotactic protein 1),fucntions to induce directional migration of mononuclear cells in the blood.However,its receptor CCR2 has been found in a variety of tissue cells(such as T cells,B cells,NK cells,neutrophils,etc.)and tumor cells.So it can directly combine with CCR2 protein and promote tumor proliferation,or recruite surrounding tissue cells involved in the formation of the tumor microenvironment.Treg cells have very important effect on the body’s immune system,and can induce immune suppression and immune escape of cancer cells in the tumor microenvironment.Many studies have shown that CCL2 in the tumor microenvironment is closely related to the recruitment of Treg cells.Our research is mainly divided into three parts:Firstly,we analyzed the IL-33 expression in clinical tissue specimens and its relationship with the clinical pathological parameters of ESCC.Secondly,we defined the effects of IL-33 on the proliferation,invasion and metastasis of ESCC cells both in-vivo and in-vitro experiments.Finally,we identified the the relationship of IL-33 as a secretory cell factor with chemokine CCL2 in tumor microenvironment and also with the immune-inhibitory Treg cells.Taken together,we tried to reveal the functional mechanism of IL-33 on the occurrence and development of ESCC.Part 1 Expression of IL-33 in patients with ESCC and its clinical significanceMethods:1.The mRNA expression levels of IL-33 in ESCC tissues and adjacent tissues were measured by Realtime PCR and its correlation with the clinical parameters of ESCC pateints was analyzed.2.The relationship between the expression level of IL-33 in ESCC tissues and the clinicopathological parameters was detected by immunohistochemistry.3.The relationship between expression level of IL-33 detected by Realtime PCR and immunohistochemistry the prognosis of ESCC patients was analyzed.Results:1.The mRNA expression level of IL-33 in ESCC was significantly higher than that of adjacent tissues.Moreover,the higher stage of ESCC is and the worse the differentiation degree is,the more IL-33 mRNA will ESCC tissue express.2.The protein level of IL-33 had a significant correlation with degree of tumor differentiation,tumor stage,the invasion ability of the tumor and survival of patients,and no correlation with patient’s age,gender and lymphatic metastasis.3.The overall survival rate of patients was low when IL-33 with the higher expression,while the survival rate was relatively high when IL-33 with the lower or no expression at ESCC,and the results were statistically significant.Summary1.IL-33 expression in the tumor tissues of ESCC patients was significantly higher than that of tissue adjacent to ESCC.The mRNA and protein expression level of IL-33 were closely related to the clinical parameters of ESCC patients,revealling that IL-33 may paly an important role in the development in ESCC.2.The expression of IL-33 in ESCC had a significant correlation with the degree of tumor differentiation,staging,invasion,and survival in patients with ESCC.The high expression of IL-33 was negatively correlated with patients’ overall survival time,thus inferred that IL-33 can be used as an indicator to predict the prognosis of patients with ESCC.Part 2 Effect of IL-33 on the biological behavior of ESCC cellsMethods:1.The IL-33 expression at mRNA level in multiple ESCC cells was detected by Realtime PCR.2.The ability of tumogeneis and invasion of IL-33-overexpressed ESCC was studied using cck-8 experiment and cloning experiment.3.The ability of invasion and metastasis of IL-33-overexpressed ESCC was studied using wound healing test and transwell experiment.4.The ability of tumogeneis of IL-33-low-erexpressed ESCC was studied using cck-8 experiment and cloning experiment.5.The ability of invasion and metastasis of IL-33-low-erexpressed ESCC was studied using wound healing test and transwell experiment.6.The effects of IL-33 overexpression on ESCC tumorgenesis were analyzed on nude mice subscutaneously injected with ESCC cells.7.The effects of IL-33-low-expression on ESCC tumorgenesis were analyzed on nude mice subscutaneously injected with ESCC cells.Results:1.The mRNA expression of IL-33 was highest in EC 109 cells,and was lowest in KYSE450 cells.So,for overexpression experiments,we choose KYSE450 cells,and for knockdown experiments,we choose EC 109 cells.2.Overexpression of IL-33 had no significant effect on proliferation of ESCC cells.3.Overexpression of IL-33 promoted the invasion and migration of ESCC cells.4.Knockdown of IL-33 gene had no significant effect on proliferation of ESCC cells.5.Knockdown of IL-33 gene restrained the invasion and migration of ESCC cells.6.Overexpression of IL-33 promoted the growth of ESCC in mouse experiments.7.Knockdown of IL-33 gene restrained the growth of ESCC in mouse experiments.Summary1.Overexpression of IL-33 or knockdown of IL-33 gene had no effect on the proliferation of ESCC,but had a significant effects on invasion and migration of ESCC cells.2.In in-vivo experiments,IL-33 had a significant effect on proliferation of ESCC.3.The different experiment results of IL-33 on ESCC proliferation between in vivo and in vitro may reveal its important role in tumor microenviroment.Part 3 Mechanism of IL-33 affecting the proliferation and invasion of ESCC cellsMethods:1.We used Cytometric Bead Array and ELISA method to test the expression of cytokine changes in IL-33-overexpressed or IL-33-knockdown ESCC cell-derived supernatant.2.Realtime PCR was used to detect the mRNA expression of IL-33 and CCL2 in the subcutaneous tumor tissues of nude mice,and the relationship between IL-33 and CCL2 was verified by analyzing data from TCGA.Exogenous IL-33 was added to ST2-knockdown cells and ST2-deficient ESCC cells,and the mRNA and protein levels of CCL2 expression were detected by Realtime PCR and ELISA.3.Western blot method was used to explore the signaling pathway of IL-33 regulating CCL2 expression in ESCC cells.4.The chemotaxis of Treg cells in microenvironment of ESCC cells were investigated by flow cytometry and transwell system.5.We used Realtime PCR methods to analyze the expression of EMT-related molecules(N-CAD,ZEB2,VIM,slug)in tumor tissue from mice to validate the mechanism of invasion and metastasis of ESCC.Results:1.CCL2 was the most significant different chemokine in the supernatant from both IL-33-overexpressed ESCC and IL-33-konckdown ESCC compared to control,suggesting that CCL2 might participate in the biological effect of IL-33.2.In the tumor tissues of overexpressing IL-33,the expression of CCL2 was also significantly increased.On the contrary,the expression of CCL2 was significantly decreased in tumor tissues with IL-33-low-expressing.Adding of recombinational exogenous IL-33 into ESCC caused a significantly elevated CCL2 expression in mRNA and protein expression.After knocking down ST2,the IL-33 receptor on cell membrance,we found that CCL2 was not significantly elevated after recombinational exogenous IL-33 adding into cell medium.The results of TCGA database analysis also showed that the expression of IL-33 was significantly positively correlated with CCL2 expression.3.The CCL2 expression decresed significantly when inhibiting the NF-κB pathway.After adding exogenous human derived IL-33,NF-κB pathway was significantly activated.When we knocked down the receptor of IL-33,ST2,NF-κB pathway activation and CCL2 expression were all inhibited with additional of exogenous IL-33.4.CCL2 and ESCC cell derived-supernatant could recruit Treg cells,and thus play a role of immune suppression in the tumor microenvironment,promoting the immune escape of ESCC,and further promoting the growth of ESCC cells.5.The expression of N-CAD,ZEB2,VIM and slug in the shIL-33 group was significantly lower than that in the NC group,and the differences were statistically significant.Summary1.The expression of IL-33 in ESCC was positively correlated with the expression of CCL2,and IL-33 could promote the expression of CCL2 through the NF-κB signaling pathway.2.CCL2 could recruit immunosuppressive Treg cells into the microenvironment of ESCC,thereby promoting immune escape of tumor cells.The mechanism by which IL-33 promoted the development of ESCC might be by increasing CCL2 expression to recruit Treg cells.3.The mechanism of IL-33 to promote the invasion and metastasis of ESCC might be related to the development of EMT,and the detailed mechanism is still required for further study.Conclusion:1.IL-33 expression in tumor tissues of patients with ESCC was significantly higher than that of tissue adjacent to ESCC,the expression of IL-33 levels was significantly associated with clinical parameters of ESCC patients.Thus IL-33 can be used as an indicator to predict the prognosis of patients with ESCC.2.IL-33 had no effect on proliferation of ESCC cells,but it significantly affected invasion and migration ability of ESCC,which may due to epithelial mesenchymal transformation:3.IL-33 could promote the secretion of CCL2 through the NF-κB signaling pathway,thus recruit Treg cells,promote the immune escape of tumors,and finally promote the development of tumor.