The Relationship And Related Mechanism Between Interleukin-35 And Progression Of Prostate Cancer

Background and purposeProstate cancer(PCa)is the second most common malignant tumor following lung cancer and the fifth leading cause of cancer-associated mortality in men worldwide.Prostate specific antigen(PSA)is the most widely used tumor marker for PCa in early diagnosis and treatment evaluation.However,PSA is not cancer specific.Benign prostatic hyperplasia(BPH),prostatic intraepithelial neoplasia,acute and chronic prostatitis,and other non-malignant prostate diseases may additionally lead to elevated plasma PSA expression levels.In addition,there is a weak correlation between PSA expression levels and PCa severity,which undermines the use of PSA in disease grading.At present,prostate biopsies are necessary;however,it is an invasive method for precise diagnosis in clinical practice.Therefore,more specific and reliable biomarkers are required for the detection,diagnosis and prognosis of PCa.Currently,patients with localized prostate cancer are usually treated by surgery or radiotherapy.However,20-40%of patients receiving radical prostatectomy and30-50%of patients receiving radiation therapy will relapse.Although ablation is very effective,it has obvious side effects.There is currently no suitable treatment for metastatic castration-resistant prostate cancer and it is associated with poor prognosis.Therefore,efforts have been made to develop more effective treatments for metastatic castration-resistant prostate cancer.In recent years,bio-immunotherapy has become a new therapy which has attracted more and more attention.Studies have shown that some tumor cells evade the recognition and killing of the immune system by activating the negative stimulus signal and regulating the inhibitory immune response.In addition,tumor cells can recruit immunosuppressive cells such as regulatory t cell(Treg)and myeloid derived inhibitory cells(MDSCs),directly or indirectly mediate the release of immunosuppressive factors,and jointly promote the occurrence and development of immunosuppressive tumor microenvironment,thus significantly promote tumor progression.Therefore,some cytokines with immunosuppressive function,such as transforming growth factor-?(TGF)-?,interleukin(IL)-6,IL-35 and so on,have attracted great attention.Immunosuppressive cytokine IL-35,a new member of IL-12 cytokine family,has become a hot research topic in the field of biological immunity of tumors.Previous studies have shown that IL-35 was highly expressed in a variety of tumor tissues,which could promote tumor angiogenesis and inhibit the anti-tumor cytotoxic lymphocyte response.In many malignant tumors,plasma IL-35 level was closely related to tumor stage,tumor size and lymph node metastasis,suggesting that IL-35might be related to the progress of tumors.The high expression of IL-35 in tumor tissues and its elevation in plasma might lead to poor prognosis of many malignant tumors.In addition,IL-35 could promote the expression of many inhibitory receptors,such as PD1,TIM3 and LAG3,thus promoting the growth of T cells in tumors.Recent studies have shown that IL-35 promotes the progression of gastric cancer by increasing Treg,MDSCs,CD3~+,CD4~+,CD8~+T cells,CD4~+memory T cells,CD8~+T cells,CD14~+monocytes and B cells in peripheral blood of patients.Overexpression of IL-35 in hepatocellular carcinoma cells could enhance apoptotic sensitivity and induce cell cycle arrest by regulating genes related to cell cycle and apoptosis,including increasing FAS expression,down-regulating cyclin D1,survivin and Bcl-2.Besides,IL-35 could induce cell proliferation and inhibit apoptosis in vitro by up-regulating cyclin B,cyclin D,CDK2,CDK4 and Bcl-2 in pancreatic cancer cells.Current clinical studies have shown that the average concentration of IL-35 in serum of patients with prostate cancer was significantly higher than that of healthy controls,suggesting that IL-35 may be involved in the occurrence of cancer.However,there is no report on the expression of IL-35 in PCa tumors and the effect of IL-35 on the progression and metastasis of PCa and its mechanism.The aim of this study was to explore the levels of IL-35 in plasma and tumor tissue of patients with PCa(PCa),to investigate the expression of IL-35 in different PCa cell lines,to investigate the effects of IL-35 on invasion,migration and proliferation of PCa cells,to detect the effect of IL-35 on tumor growth and survival rate of mice bearing subcutaneous PCa,and to investigate the effect of IL-35 on proliferation of PCa cells and tissue angiogenesis of PCa in mice,and the effect on immune function.We explored the role of IL-35 in the carcinogenesis and development of PCa from the clinical level,cell level in vitro and experimental level in vivo,and investigated related regulation mechanism.Methods1.Plasma IL-35 levels in patients with PCa(pca)were measured by enzyme-linked immunosorbent assay(ELISA).The correlation between plasma IL-35 levels and clinical parameters and pathological features was analyzed.The clinical role of IL-35in the diagnosis of PCa was established by using the receiver operating characteristic curve(ROC),).Survival curve was used to compare the overall survival rate of PCa patients with different IL-35 levels.Human PCa tissue microarray was used to detect the expression of IL-35 in human PCa tissue,and the relationship between the expression of two subunits of IL-35 and clinical pathology was analyzed.2.The expression of two subunits of IL-35 in three human PCa cell lines(LNCaP?DU145?PC-3)and mouse PCa cell line RM-1 was detected by Western blot and RT-PCR.PCa cells cultured in vitro were divided into different groups.The invasive ability of the cells in each group was detected by transwell chambers,and the migration ability of the cells in different treatment groups was detected by wound healing assay.The proliferation ability of the cells in each group was detected by CCK-8 kit.3.The PCa model of RM-1 mice was constructed.The normal control group,the IL-35 monoclonal antibody group and the IL-35 neutralizing antibody group were set up respectively.The gross observation was carried out,the growth curve of tumor and the survival curve of mice were drawn.The changes of tumor volume and survival rate were analyzed.Plasma IL-35 level was measured by ELISA.The expression of two subunits of IL-35(EBI3 and p35),angiogenesis index(CD31)and cell proliferation index(Ki67)were detected in tumor tissue by immunohistochemistry andv Western blot.The lung tissue was stained with HE to calculate the lung metastasis rate in each group.The percentages of MDSCs,Treg,CD4~+T,CD8~+T cells in the blood and spleen of each group were measured by flow cytometry.Results1.The plasma IL-35 concentration in patients with PCa was significantly higher than that in patients with benign prostatic hyperplasia group and healthy control group.The plasma IL-35 concentration in patients with PCa increased with the progression of tumor stage and was positively correlated with Gleason score.The plasma IL-35 concentrations in patients with local lymph node metastasis and distant metastasis were higher than those without local lymph node metastasis and distant metastasis,respectively.The overall survival rate in patients with high plasma IL-35concentration was significantly lower than that in patients with low concentration.IL-35 was more effective than PSA in the diagnosis of lymph node metastasis and distant metastasis in patients with PCa.2.The expression of two subunits of IL-35,EBI3 and p35,in PCa tissues was significantly higher than that in adjacent normal tissues.The expression of EBI3 and p35 increased with the increase of Gleason score.3.The expression of EBI3 and p35 protein was high in PC-3 cell line and RM-1cell line,which were with highly metastatic potential,while the expression level of EBI3 in DU145 cell line and LNCap cell line with moderate metastatic potential was high but p35 was relatively low.Accordingly,mRNA levels of EBI3 and p35 was highly in PC-3 and RM-1 cell lines,while in DU145 and LNCap cell lines,the mRNA of EBI3 was higher and p35 was relatively low.4.Compared with the control group,IL-35 significantly increased the number of RM-1 cells in the lower side of transwell chamber,the coverage area of PCa cells in wound healing assay and the OD value in CCK-8 assay.However,number of RM-1cells in the lower side of transwell chamber,the coverage area of PCa cells and the OD value in IL-35 neutralizing antibody group were significantly lower than those in scramble group.5.IL-35 increased the volume of PCa,increased the lung metastasis rate,and reduced the overall survival rate of mouses.The expression of CD31 and Ki67 in IL-35 group was higher than control group,but the expression of CD31 and Ki67 in IL-35 neutralizing antibody was decreased in PCa tissue than scramble group.6.Il-35 increased the proportion of regulatory T cells(Treg)and myeloid-derived inhibitory cells(MDSCs)in tumor microenvironment,and decreased the proportion of CD4~+and CD8~+T cells.Conclusion1.IL-35 was highly expressed in plasma and tissues of patients with PCa.It was positively correlated with the occurrence,progression,metastasis and poor prognosis of PCa.It could be used as a biomarker for the development of PCa.2.IL-35 could promote the invasion,migration and proliferation of PCa cells,and neutralizing IL-35 could significantly inhibit the invasion,migration and proliferation of PCa cells.3.IL-35 was associated with progression,metastasis and poor prognosis of PCa in mouse.IL-35 could be used as a target for the treatment of PCa.4.IL-35 promoted tumor growth by up-regulating CD31 and Ki67 to promote angiogenesis and cell proliferation of PCa in mouse.Neutralizing IL-35 therapy could inhibit angiogenesis and cell proliferation of PCa in mice and thus played an anti-PCa role.5.IL-35 could up-regulate CD4~+and CD8~+T cells by up-regulating Treg cells and MDSCs,so as to achieve immunosuppressive effect and promote malignant progression of PCa.