Expression And Clinical Significance Of NKG2D And Its Ligand MICA In Patients With Prostate Cancer

BackgroundProstate cancer is the second most common cancer worldwide and the fifth leading cause of cancer-related deaths in men.Screening for prostate cancer with serum prostate-specific antigen(PSA)is designed to detect prostate cancer at an early,interentable stage for treatment and to reduce overall and disease-specific mortality.But a recent retrospective study found that PSA screening led to an increase in prostate cancer diagnoses,but did not reduce overall or disease-specific mortality.Instead,screening for prostate cancer remains controversial because it may increase the risk of complications from overdiagnosis and treatment.Therefore,additional biomarkers are needed to assist in the diagnosis,treatment and prognosis of prostate cancer.Cytotoxic lymphocytes are key players of immune surveillance as they have the ability to selectively kill malignant or infected cells.These lymphocytes include natural killer(NK)and T cells which share the expression of the activatory Natural killer group 2,member D(NKG2D)receptor which stimulates effector responses upon engaging various stressinducible ligands,hereafter called NKG2D ligands(NKG2DLs).The NKG2D-NKG2DL interaction is therefore a key event in the regulation of both innate and adaptive immune responses.Upon ligand recognition,NK and T cells harboring NKG2D are activated to trigger lysis of the target cell.The binding of the receptor to its ligand also triggers cytokine and chemokine production,e.g.INF-y,which modulate immune response and help maintain tissue homeostasis.MICA and MICB are stress-induced molecules recognized by NKG2D,one of major activation receptors of natural killer(NK)cells.Upon binding to NKG2D,NKG2D-mediated cytolytic immune response of immune effector cells will be activated against virally infected and MICA expressing of tumor cells.In the early oncogenic development,membrane-bound MICA serves as a key signal to recruit anti-tumor immune effectors.Nevertheless,both MICA polymorphic features and its dysregulated expression in evolving tumors have resulted in tumor evasion in various cancer types.Therefore,it is of great significance that we understand MICA genetics,polymorphisms,mechanisms of MICA-associated tumor escape and molecular/cellular modulation of MICA for the diagnosis,treatment and reconstruction of tumor immune monitoring.Part I The prognostic value of serum soluble MICA(sMICA)level in patients with prostate cancerObjectiveBy evaluating the serum soluble MICA(sMICA)level,PSA level,PS score,EOD score and tumor pathology of patients with prostate cancer in different stages,the prognostic factors of patients with prostate cancer were identified.MethodsSerum sMICA level was detected in 136 prostate cancer patients,including 36 of stage B,20 of stage C and 80 of stage D.Univariate and multivariate Cox proportional risk models were used to assess the prognostic significance with tumor histology,physical condition score(PS),bone metastasis,serum prostate-specific antigen(PSA)levels,and sMICA levels for disease-specific survival.Results1.The sMICA levels of patients with stage B,C and D prostate cancer were 198.4±7.2 pg/ml,229.8±8.3 pg/ml and 578.4±12.2 pg/ml.There was no significant difference in serum sMICA level between stage B and stage C(P>0.05),and there was significant difference between stage B and D,C and D(P<0.05).2.Serum PSA levels of stage B,C and D prostate cancer patients were 63.3±13.7 ng/ml,178.1±14.6 ng/ml and 303.8±157.1 ng/ml,respectively,with statistically significant differences(P<0.05).SMICA was significantly correlated with PSA(r=0.76,P<0.05).3.SMICA has auxiliary diagnostic value(AUC=0.92),the Youden index corresponding to the optimal critical point is 283 pg/ml,the corresponding sensitivity is 85.3%,and the specificity is 83.3%.4.Univariate analysis showed that the survival rates of patients with EOD?1,sMICA>283 pg/ml,PS>1 and PSA>100 ng/ml were significantly lower than their corresponding items(P<0.05),and there was no significant correlation between tumor histology and prognosis.Multivariate Cox regression analysis showed that sMICA and EOD were important factors affecting the survival time of prostate cancer.Conclusion1.The serum sMICA level of patients with distant metastatic prostate cancer(stage D)was significantly higher than that of patients with locally infiltrating prostate cancer(stage C)and localized prostate cancer(stage B).2.Serum sMICA level is positively correlated with PSA level in patients with prostate cancer.3.SMICA is of auxiliary diagnostic value in prostate cancer.4.SMICA and EOD are important factors affecting the survival time of prostate cancer.Part II Correlation between the expression of NK cell activated receptor NKG2D and its ligand MICA in patients with prostate cancerObjectiveBy detecting the expression level of NK cell surface activated receptor NKG2D and its ligand MICA in peripheral blood of patients with prostate cancer,the correlation between the expression of NK cell surface activated receptor NKG2D and its ligand MICA and the relationship between NKG2D-sMICA and immune escape from prostate cancer and its clinical significance were discussed.MethodsThe levels of soluble MICA(sMICA)in serum and NK receptor NKG2D in peripheral blood of 60 patients with prostate cancer and 20 healthy volunteers were detected by ELISA and flow cytometry before and after treatment,respectively.Results1.Expression level of sMIC AThe serum sMICA concentrations of the prostate cancer group and the normal group were 290.1±86.1 pg/ml and 109.9±15.5 pg/ml,respectively,with statistically significant differences.Among the prostate cancer patients,22 patients of stage B,17 patients of stage C,and 21 patients of stage D had serum sMICA concentrations of 199.8±14.9 pg/ml,283.8±26.7 pg/ml,and 389.8±40.6 pg/ml,respectively,which were significantly higher in stage D than in stage B and C,with statistically significant differences(P<0.05).2.NKG2D expression levelThe expression levels of NKG2D in the prostate cancer group and the normal group were 78.9±3.8%and 90.4±4.0%,respectively,with statistically significant differences.The expressions of stage B,C and D NKG2D in prostate cancer patients were 81.0±4.8%,76.3±4.0%and 67.0±4.0%,respectively,with statistically significant differences(P<0.05).3.Correlation between NKG2D and sMICA in patients with prostate cancerSpearman correlation coefficient was-0.896,showing negative correlation and statistical significance(P<0.05).4.SMICA and NKGD after treatmentThree months after radical prostatectomy or androgen deprivation treatment,among the prostate cancer patients,the serum sMICA concentrations were 138.2±15.4 pg/ml,215.9±18.7 pg/ml and 304.8.6±35.2 pg/ml in 17 cases of stage B,22 cases of stage C and 21 cases of stage D,respectively.The expression of stage B,C and D NKG2D was 88.2±4.9%,81.8±4.0%and 77.2±4.7%,respectively,in the patients with prostate cancer after the operation,and the difference was statistically significant(P<0.05).Conclusion1.The expression of NKG2D is decreased in prostate cancer patients,which leads to the decreased activity of NK cells to kill tumors,which may be an important mechanism to cause immune escape of prostate cancer.2.With the progress of prostate cancer,MICA is removed from the surface of prostate cancer to form sMICA,and the content of sMICA in serum is increased.However,the expression of NK cell receptor NKG2D was down-regulated.There was a negative correlation between serum sMICA level and NKG2D expression of activated NK cell receptor in peripheral blood of prostate cancer patients.The abnormal increase of sMICA may be an important reason for the decrease of NKG2D expression.3.Radical prostatectomy and androgen deprivation therapy can reduce serum sMICA levels and increased the expression of NKG2D.Part ? Effects of recombinant sMICA protein and gemcitabine on NK cell functionObjectiveTo study the effect of recombinant human sMICA and chemotherapeutic drug gemcitabine on NK cell activated receptor NKG2D and its secretion of IFN-y in vitro.MethodsPeripheral blood mononuclear cells(PBMNC)were isolated by density gradient centrifugation,and NK cells were separated by immune magnetic beads.NK cells were divided into blank control group,sMICA group and GEM group.Blank control group to add volume of complete medium.SMICA group added different concentrations of recombine sMICA(250 pg/ml,500 pg/ml and 1000 pg/ml),respectively.In the GEM group,recombinant sMICA(250 pg/ml,500 pg/ml,1000 pg/ml)and gemcitabine(5ug/ml)were added respectively.After overnight incubation with NK cells,NK cells were collected and the expression level of NKG2D receptor was detected by flow cytometry.Culture medium supernatant was collected,and IFN-y level was detected by ELISA kit.Results1.The expression rate of NKG2DThe expression rates of NKG2D in sMICA1 group,sMICA2 group and sMICA3 group were 74.1±5.4%,57.83±3.9%and 41.1±5.4%,respectively,which were significantly lower than that in the blank control group 90.6±4.0%,with statistically significant differences(P<0.05).With the increase of sMICA stimulation concentration,the expression rate of NKG2D receptor in NK cells decreased gradually,and the difference between groups with different concentrations was statistically significant(P<0.05).The expression rates of NKG2D in GEM1 group,GEM2 group and GEM3 group were 85.5±2.9%,66.3±5.0%and 51.8±5.6%,respectively,with statistically significant differences(P<0.05).The expression levels of NKG2D in smical-geml,smica2-gem2 and smica3-gem3 groups were compared in pairs(%),and the differences were statistically significant(P<0.05).2.The concentration of IFN-? in supernatant of culture solutionThe concentration of IFN-? in group sMICA1,sMICA2 and sMICA3 was 190.5±12.4 pg/ml,100.4±10.5 pg/ml and 61±6.4 pg/ml,respectively,which were significantly lower than that of the blank control group 307.3±16.6 pg/ml,and the difference was statistically significant(P<0.05).With the increase of sMICA stimulation concentration in the experimental group,the secretion level of IFN-y in NK cells decreased gradually,and the difference was statistically significant(P<0.05).The concentration of IFN-y in group GEM1,GEM2 and GEM3 were 216.3±18.8 pg/ml,161.8±11.1 pg/ml and 93.3±18.5 pg/ml,respectively,with statistically significant differences among the groups(P<0.05).Pair comparison of concentration of IFN-y in smical-geml,smica2-gem2 and smica3-gem3 groups showed statistically significant differences(P<0.05).Conclusion1.Recombinant soluble MICA(sMICA)can down-regulate the expression of NKG2D receptor in NK cells.2.Recombinant soluble MICA(sMICA)can reduce the level of IFN-? secreted by NK cells.3.Gemcitabine has immunoregulatory effects,and can improve the function of NK cells by up-regulating the expression of NKG2D.NK cells combined with gemcitabine in the treatment of advanced tumors deserve further study.