The Mechanism Of Sulfatase 2 Regulating The Polarization Of Macrophages In The Microenvironment Of Bladder Cancer

Background and Aims: Bladder cancer(Bca)is one of the most common malignant tumors in urinary tract tumors.Its incidence rate and mortality rate ranks first,which brings serious threats and challenges to the survival and quality of life of patients.Bladder cancer can be divided into non-muscular invasive bladder cancer(NMIBC)and muscular invasive bladder cancer(MIBC).Non-muscular invasive bladder cancer is characterized by easy recurrence,and muscular invasive bladder cancer is characterized by high malignancy,easy metastasis and poor prognosis.However,in the past 20 years,there has been no significant progress in the treatment of MIBC.The current treatment for bladder cancer is still radical cystectomy and platinum-based chemotherapy,which has limited efficacy and a low 5-year survival rate for patients.Therefore,we urgently need to further discover and study the key molecules and key mechanisms involved in the malignant progression of bladder cancer.In the past five years,with the development of immunotherapy,the treatment of bladder cancer has entered a new stage,and the research on bladder cancer immune checkpoints and immune microenvironment has gradually deepened.Tumor cells secrete various immunosuppressive factors(such as TGF-β,IL-6,etc.)and antiapoptotic factors(such as PGE2,VEGF,etc.)to recruit and differentiate immune cells with immunosuppressive phenotypes,such as Myeloid derived suppressor cells(MDSC),tolerogenic dendritic cells(t DC),regulatory T cells and M2 type macrophages from the same source,creating a highly tolerant microenvironment.Macrophages are heterogeneous cells that can polarize cell populations with unique phenotypes and functions in different microenvironments.The polarization of macrophages can be divided into M1 type(classical activated Macrophages),M2 type(alternatively activated macrophages).Studies have confirmed that M2 macrophages can exert immunosuppressive functions and affect tumor progression and metastasis.The previous data showed that sulfatase 2(sulf2)was highly expressed in bladder cancer and was positively correlated with the expression of M2 macrophages.Sulfatase 2(SULF2)is a member of the sulfatase family.It plays an important role in cancer progression by modifying the sulfate pattern of heparan sulfate proteoglycan(HSPG)on the surface of most cancer cells.SULF2 plays an important role in cancer progression.However,So far,the regulatory mechanism of sulf2 in the tumor microenvironment of bladder cancer is not clear.This study hopes to provide a theoretical basis for treatment of bladder cancer by exploring the molecular mechanism of sulf2 in regulating macrophage polarization in the tumor microenvironment of bladder cancer.Methods:1.First,analyze the expression and prognosis of SULF2 in bladder cancer patients and normal controls through the Oncomine database and the data of203 samples from our center.Secondly,through quantitative real time-PCR(q PCR),western blot(western blot,WB)and immunohistochemical(immunohistochemical,IHC)experiments to further detect and analyze the expression of SULF2 in bladder cancer tissues and adjacent tissues Happening.Finally,we also verified the expression levels of SULF2 in bladder cancer cell lines(T24,UMUC-3,and J82)and immortalized urothelial cells(SV-HUC-1)through q PCR and WB experiments.2.Select the cell line T24 with the highest expression of SULF2 to construct a cell line with stable SULF2 knockdown(sh-SULF2),and detect the knockdown efficiency of SULF2 by real-time fluorescent quantitative PCR and Western blotting.The cell line UMUC-3 with low expression of SULF2 was selected to construct a cell line with stable and high expression of SULF2 gene(oe-SULF2),and real-time fluorescent quantitative PCR and western blotting were used to detect the overexpression efficiency of SULF2.The bladder cancer cells with stable knockdown/overexpression of SULF2 and the control cell line(Negative Control)were injected subcutaneously into nude mice,and the ability of the two groups of cells to form tumors subcutaneously was compared.3.CCK8 experiment,plate cloning and other experiments are used to detect the effect of SULF2 on the proliferation ability of T24 cells and UMUC-3 cells;the Transwell chamber is used to detect the effect of SULF2 on the migration and invasion of T24 and UMUC3 stable cells,flow cytometry Cell analysis is used to detect the changes of SULF2 on the cell cycle and apoptosis of T24 cells and UMUC-3 cells.4.TCGA database analyzes the expression of different immune cells in the microenvironment of bladder cancer.The group of immune cells with the largest expression difference,namely macrophages,was screened out.The mouse bladder cancer cell line MB49 was used to construct a mouse SULF2 gene stable knockdown cell line(sh-SULF2),and real-time fluorescent quantitative PCR was used to detect the knockdown efficiency of SULF2.The MB49 cell line was used to construct a C57 mouse bladder tumor model in situ,and flow cytometry was used to analyze the immune microenvironment in the mouse bladder tumor in situ.In vivo experiments,a co-culture model of tumor cells and macrophages was constructed,q PCR and WB were used to detect the polarized markers of macrophages(CD163,CD206,etc.).5.Elisa analysis of the changes in the factors secreted by tumor cells after knockdown or overexpression,the most different cytokine IL-8 was selected,and the analysis found that SULF2 affects the IL-8 signaling pathway and inhibits its key genes.6.WB experiment to detect the changes of JNK2/STAT3 pathway.Furthermore,STAT3 inhibitors and agonists were used to inhibit or activate the expression of STAT3 phosphorylation,and further analyze the influence of STAT3 pathway on SULF2 regulation of macrophage polarization.Results: 1.Data analysis of the TCGA database and 203 samples from our center found that SULF2 is highly expressed in bladder cancer and is closely related to T stage,tumor diameter,lymphatic metastasis,etc.It is an independent predictor of prognosis.Immunohistochemistry showed that the expression of SULF2 was significantly higher than that of adjacent tissues.For bladder cancer cell lines,through q PCR and Western Blot,it was found that the expression of SULF2 in T24,UMUC3 and J82 was significantly higher than that of immortalized normal bladder epithelial cells SV-HUC-1.2.Successfully constructed a stable transgenic cell sh-SULF2 that knocked down SULF2 in T24 cells,and constructed a stable transgenic cell oe-SULF2 that overexpressed SULF2 in UMUC3 cells.Through CCK8,it was found that the proliferation ability of sh-SULF2 was significantly decreased,and the proliferation ability of oe-SULF2 was significantly enhanced.Through the Transwell chamber experiment,it was found that the migration and invasion ability of sh-SULF2 was significantly decreased,and the migration and invasion ability of oe-SULF2 was significantly enhanced.Western Blot showed significant changes in EMT-related indicators.The cell cycle showed that: knockdown or overexpression of SULF2 resulted in G1 phase block.Flow cytometry analysis found that apoptosis increased significantly after knocking down SULF2.3.Through the analysis of the TCGA database,we found that in the microenvironment of bladder cancer,the infiltration of macrophages increased significantly.Further analysis we found that SULF2 is significantly related to M2 macrophages.We successfully constructed an orthotopic model of bladder cancer in mouse MB49 cell line,and flow cytometry found that M2 macrophages in the SULF2 knockdown group were significantly reduced.After co-culturing bladder cancer cells with macrophages THP1,it was found that CD163 and CD206 in the sh-SULF2 group were significantly reduced,while CD163 and CD206 in the oe-SULF2 group were significantly increased4.Through screening of multi-factor,we found that the expression of IL-8 is significantly different after knocking down or overexpressing SULF2,and SULF2 has a significant positive correlation with IL-8.Furthermore,we found that after adding IL-8 or IL-8 inhibitor to THP1 cells,both CD163 and CD206 changed significantly.At the same time,when IL-8 was added to the co-cultivation system,we also found that CD163 and CD206 also changed to varying degrees.We found that SULF2 can regulate the expression of IL-8 through β-catenin in bladder cancer cells.We constructed a cell line that knocked down β-catenin.We found that after co-culture with THP1,after knocking down β-catenin,CD163 and CD206 Both dropped significantly.5.We co-cultured bladder cancer cells overexpressing or knocking down SULF2 with THP1,and we found that the phosphorylation of JAK2 and STAT3 on the STAT3 pathway changed significantly.When we added IL-8 exogenously to THP1 cells,we found that the phosphorylation of JAK2 and STAT3 changed significantly.While we used STAT3 inhibitors in the co-culture system,we found that the expression of CD206 and CD163 was inhibited.Conclusion: 1.SULF2 is significantly increased in bladder cancer tissues and bladder cancer cell lines,and its expression is closely related to clinicopathological factors of bladder cancer.The expression of SULF2 is an independent predictor that affects the prognosis of bladder cancer.2.Knockdown or overexpression of SULF2 will affect the biological functions of bladder cancer,such as proliferation,migration,invasion and so on.3.Mouse orthotopic tumorigenesis cells found that after SULF2 knockdown,M2 type macrophages were significantly reduced.After knocking down SULF2,in vitro co-culture experiments also confirmed that the expression of CD206 and CD163 was significantly reduced,while THP1 and overexpressing SULF2 cells After the line was cultured,the expression of CD206 and CD163 was up-regulated.4.q PCR verified that the IL-8 secreted by bladder cancer was significantly changed after knocking down or overexpressing SULF2.Further experiments confirmed that SULF2 can regulate the secretion of IL-8 through β-catenin.5.IL-8 may affect the transcription of CD206 and CD163 by affecting the JAK2/STAT3 signaling pathway of macrophages.