Regulatory T-cell-specific Signature Predict Prostate Cancer Prognosis And Immune Response

Objective: Prostate cancer is a tumor with significant immunosuppressive microenvironment,with high malignancy and easy recurrence.Regulatory T cells(Tregs)are the key cells that play an immunosuppressive role in tumor microenvironment.Therefore,identifying specific biomarkers of Tregs is of great significance in improving the survival rate of prostate cancer patients.Traditional Tregs quantitative methods often have bias and instability,and the commonly used prostate cancer biopsy and puncture detection method in clinical practice is complex and has a certain degree of trauma.Therefore,this study intends to use the public data set of purified immune cells from peripheral blood to identify Tregs specific mRNAs based on bioinformatics methods,develop and verify the risk characteristics of Tregs,and provide a non-invasive test basis for accurately predicting the prognosis and immune response of prostate cancer.Methods:1.Using the GEO dataset of Tregs and 19 other immune cells,identify Tregs specific mRNAs through differential expression analysis,and develop and validate the risk characteristics of Tregs based on univariate and multivariate COX analysis methods..In TCGA and ICGC data sets,Kaplan-Meier survival curve and ROC curve were used to evaluate the predictive ability of the risk characteristics of Tregs to the prognosis of prostate cancer,and the correlation analysis of clinicopathologic factors and chi-square test were used to explore the relationship between the risk characteristics and clinical characteristics of Tregs.2.The ESTIMATE algorithm,CIBERSORT algorithm,ss GSEA algorithm and Immune Cell AI web analytics tool were used to comprehensively evaluate the tumor immune score and the infiltration of tumor immune cells,analyze the correlation between the risk characteristics of Tregs and tumor immunity,and further analyze its predictive ability to the degree of tumor infiltration of Tregs through principal component analysis and ROC curve.3.The relationship between Tregs risk characteristics and tumor immunotherapy response was analyzed through five urinary system tumor immunotherapy response data sets including prostate cancer,and the predictive ability of Tregs risk characteristics on tumor immunotherapy response was evaluated through ROC curve.The GSCALite web analysis tool analyzes the genetic mutations and methylation differences of five genes in Tregs risk characteristics.4.Tregs and effector T cells(Teffs)were extracted from the peripheral blood of patients with prostate cancer and healthy volunteers by gradient centrifugation and magnetic bead separation using Tregs separation kit for amplification and culture,and the cell surface markers were verified by flow cytometry.The protein expression of Tregs prognostic characteristic gene in prostate cancer patients and healthy volunteers was determined by Western blot.After using si RNA to silence the prognostic characteristic genes in Tregs,the effect of gene silenced Tregs on the proliferation of Teffs was detected by CCK-8experiment.Results:1.A prognostic feature consisting of five Tregs specific mRNAs(SOCS2,EGR1,RRM2,TPP1 and C11orf54)was constructed.This prognostic feature is superior to the traditional clinical parameters-prostate specific antigen(PSA)level,Gleason score and clinical TMN stage in predicting the RFS of prostate cancer patients.Kaplan-Meier survival curve analysis and chi-square test of clinicopathologic factors showed that patients in the high-risk group were more likely to relapse and had a higher recurrence rate than those in the low-risk group(P<0.01).At the same time,univariate and multivariate COX regression analysis also found that risk score was significantly correlated with RFS(P<0.001)and was a risk factor for prostate cancer.2.In the analysis of immune score of prostate cancer,it was found that the high-risk group had higher immune score(P<0.005)and lower tumor purity(P<0.05),and the predictive ability of risk score on tumor immune score and cytolytic activity(CYT)was better than the classic parameters such as tumor mutation burden(TMB),glycolytic activity,PSA level,Gleason score and clinical TMN stage.The results of tumor immune cell infiltration analysis showed that the degree of tumor infiltration of Tregs in high-risk group was significantly higher(P<0.05),and the predictive ability of risk score on tumor infiltration of Tregs was maybe higher than that of Fox P3,the classic index of Tregs.3.The analysis of immune response of different cancer patients showed that the patients with high risk of bladder urothelial carcinoma,renal clear cell carcinoma,prostate cancer and renal cell carcinoma had a higher response rate to immunotherapy,and the risk score was positively correlated with the gene expression of most immune checkpoints(P<0.05).The genetic variation analysis of GSCALite showed the mutations and methylation differences of five genes in the risk characteristics of Tregs.The results showed that each of the five genes had single-nucleotide mutations and copy number mutations in different degrees,and there were significant methylation differences between normal and prostate cancer samples(P<0.001).4.Through Western blot analysis,it was found that the expression of RRM2 in the Tregs of prostate cancer patients was higher than that in the peripheral blood of normal volunteers(P<0.05),while the expression of SOSC2,ERG1,TPP1 and C11orf54 was lower(P<0.05).The results were consistent with the prognosis of Tregs,indicating that RRM2 might be a risk factor for prostate cancer.We also successfully constructed the Tregs model of silencing RRM2.Through the CCK-8 proliferation experiment,we found that under the co-culture conditions of different cell ratios,si-RRM2 Tregs inhibited the proliferation of Teffs significantly lower than Tregs(P<0.001),indicating that RRM2 played an important role in the inhibition of Teffs proliferation by Tregs.Conclusion: In this study,a risk assessment model of Tregs risk characteristics was constructed,which can predict the prognosis,immunotherapy and drug resistance of prostate cancer,and provide a new way to realize direct,non-invasive and inexpensive detection of prostate cancer.