Effect Of CD73 On The Proliferation Of Gastric Cancer Cells And Its Molecular Mechanism

[Background and objective]Globally,gastric cancer(GC)is one of the most common cancers.In China,GC ranks second in the incidence of malignant tumors and the third in the cancer-related mortality.Although the improvement and breakthrough of surgery,radiotherapy,chemotherapy and targeted therapy and the survival rate of GC patients after treatment has improved to some extent in recent years,the5-year survival rate is still low.Thus actively seeking for new GC molecular markers and therapeutic targets,to improve the early diagnosis and treatment effect now is the focus of the gastric cancer research.CD73 is a multifunctional transmembrane glycoprotein encoded by NT5 E gene and anchored on the surface of cell membrane by glycosylphosphatidylinositol(GPI).It is one of the pivotal rate-limiting enzymes in the extracellular purine metabolic pathway.Recent studies found that CD73 has two functions,one is extracellular hydrolysis enzyme and the other is non-hydrolysis enzyme.The hydrolysis enzyme of CD73 takes part in regulating tumor cell proliferation,angiogenesis,immune escape,and inhibiting immune response.The nonhydrolysis enzyme of CD73 is involved in adjusting the invasion and metastasisof cancer cells.Researches found that CD73 expression levels were abnormal in breast,colorectal,head and neck squamous cell,stomach and liver cancers.However,the potential role and mechanism of CD73 in the development of GC have not yet been clarified.In this study,GC cell lines were served as experimental object.Firstly,we research the effect of APCP,a CD73 specific inhibitor,on GC BGC-823 cells proliferation in vitro and its possible mechanism by applying CD73 hydrolysis enzyme activity detection,Western Blot,Real time quantitative PCR,CCK-8and flow cytometry.Secondly,by constructing lentivirus vector shNT5 E and transfecting BGC-823 cells,then research the effect of LV-shNT5 E in vivo and in vitro on cells proliferation and its potential mechanism.Finally,the expression of CD73 in TCGA gastric cancer database was analyzed to explore its clinical significance,providing the basis and direction for the study of the role and mechanism of CD73 in the occurrence and development of GC.[Methods]1、Effect of CD73 specific inhibitor APCP on GC cells proliferationThe CD73 hydrolysis enzyme activity detection,Western blot and Real time quantitative PCR were used to determined the CD73 hydrolysis enzyme activity,protein and NT5 E mRNA expression levels in 4 GC cells lines and GES-1 cells.APCP was applied to treat BGC-823 cells,hydrolysis enzyme activity detection was used to determine the effect of APCP on cells hydrolysis enzyme activity,CCK-8 and colony formation experiments were used to observe the effect of APCP on cell proliferation,flow cytometry was used to detect the effect of APCP on apoptosis and cell cycle,and Western Blot was used to detect the effect of APCP on CD73 and EGFR/PI3 K expression.2、Effect of lentiviral mediated shNT5 E on GC cells proliferationLentivirus vector shRNA targeting NT5 E was constructed and then transfected to BGC-823 cells,the expression of EGFP in cells was observed by fluorescence microscopy to determine the transfection efficiency.Real time quantitative PCR was used to determine the sequence with the best interference efficiency after LV-shNT5 E transfected to BGC-823 cells.CCK-8 and colony formation experiment were used to observe the effect of LV-shNT5E-1 on cell proliferation.Flow cytometry was used to detect the effect of LV-shNT5E-1 on cell apoptosis and cell cycle.Puromycin was used to screen and construct BGC-823 cells with stably LV-shNT5E-1 transfected.The nude mice subcutaneous tumorigenesis experiment was applied to observe the effect of LV-shNT5E-1 on the tumor proliferation in vivo and the expression of proliferation marker Ki67.Hydrolysis enzyme activity detection was used to determine the effect of LVshNT5E-1 on cells hydrolysis enzyme activity.The effect of LV-shNT5E-1 on the expression of CD73 and EGFR/PI3 K was determined by Western blot.3 、 The expression and clinical significance of CD73 in GC were analyzed based on TCGA databaseThe expression data of CD73 in 328 GC patients with complete clinicopathological and follow-up data were acquired from the TCGA database.CD73 expression differences between GC and adjacent normal tissues were compared.25 pairs matched GC and adjacent normal tissues were selected from328 GC patients,and CD73 expression differences was analyzed in these paired tissue.Single factor Chi-square test was used to analyze the correlation between CD73 expression level and clinicopathological data in 328 GC patients.The correlation between CD73 expression level and patient survival was analyzed by Kaplan-meier method.Univariate and multivariate regression analysis determined whether CD73 was a risk factor for prognosis of GC patients or not.[Results]1 、 The activity of hydrolysis enzyme,NT5 E mRNA and protein expression in BGC-823 cells were significantly higher than that of GES-1(P<0.01),the activity of hydrolysis enzyme,NT5 E mRNA and protein expression in MGC-803 and SGC-7901 cells were not significantly different from that of GES-1(P> 0.05),and the activity of hydrolysis enzyme,NT5 E mRNA and protein expression in AGS cells were significantly lower than that of GES-1(P<0.05).The results of hydrolysis enzyme activity detection indicated that APCP could significantly inhibit the enzyme activity of BGC-823 cells in a dose-dependent manner(P<0.05).CCK-8 and colony formation experiments suggested that APCP could significantly inhibit the cells proliferation and colony formation in a dose-dependent manner(P<0.05).Flow cytometry suggested that APCP could induce apoptosis,mainly in the early stage apoptosis(P<0.01).Cell cycle experiments suggested that APCP could arrest cell cycle in G0/G1 phase(P<0.01).Western blot indicated that APCP could promote the expression of CD73 whereas inhibit the EGFR/PI3 K expression(P<0.05).2、Lentivirus vector shRNA targeting NT5 E was successfully constructed and transfected to BGC-823 cells.Real time quantitative PCR showed that LVshNT5 E significantly inhibited the NT5 E expression compared with LV-shNC group,and the LV-shNT5E-1 has the best interference efficiency(P<0.005).The results of CCK-8 and colony formation experiment showed that LV-shNT5E-1significantly promoted cells proliferation and clone formation compared with LV-shNC(P<0.01).The results of flow cytometry suggested that the apoptosis rate of LV-shNT5E-1 group was not significantly changed compared with LVshNC group(P> 0.05).Cell cycle experiment showed that the proportion of Sphase cells in LV-shNT5E-1 group was significantly higher than that in LV-shNC group(P< 0.01).The results of nude mice subcutaneous tumor experiment showed that the growth rate and the volume of tumor in LV-shNT5E-1 group has no significance compared with that in LV-shNC group(P>0.05).IHC results showed that the expression of CD73 was significantly decreased in the LVshNT5E-1 group(P<0.05),while the expression of Ki67 was significantly increased(P<0.01).Hydrolysis enzyme activity assay showed that compared with LV-shNC group,LV-shNT5E-1 group had significantly enhanced hydrolysis enzyme activity(P<0.01).Western blot indicated that LV-shNT5E-1could inhibit CD73 expression and promote the expression of EGFR/PI3K(P<0.05).3、We found that the expression of CD73 in the GC samples(328 cases)was higher than that in the adjacent normal tissues(25 cases)(P< 0.01)when analyzed data of TCGA database.CD73 expression in GC was significantly higher than that in the adjacent tissues in 18 of total 25 pairs matched samples(P<0.01).Correlation analysis between CD73 expression level and clinicopathological characteristics showed that high CD73 expression was significantly correlated with AJCC clinical stage(P=0.003),T stage(P=0.038),and N stage(P< 0.001),whereas not with age(P=0.062),gender(P=0.08),tumor grade(P=0.392),and M stage(P=0.432).Survival analysis showed that the overall survival rate of patients with high CD73 expression was significantly lower than those with low expression(HR=1.507,log-rank P< 0.05).Univariate Cox regression analysis showed that clinical stage(P=0.001),T stage(P=0.041),N stage(P=0.009),and CD73 expression(P=0.027)were significantly associated with increased risk of death.Multivariate Cox regression analysis showed that high expression of CD73(HR=1.443,95%CI=0.996-2.09,P=0.046)and clinical stage(HR=1.404,95%CI =1.009-1.952,P=0.044)can be used as an independent risk factor for the prognosis of GC.[Conclusion]1 、 The hydrolysis enzyme activity of CD73 in GC cells was positively correlated with the expression of CD73.The higher the expression of CD73,the stronger the hydrolysis enzyme activity.The hydrolysis enzyme activity and the expression level of CD73 were significantly different between different GC cell lines.2、 APCP can inhibit the enzyme activity and proliferation of BGC-823 cells,while LV-shNT5 E may promote the enzyme activity and proliferation of the cell.The mechanism of this process may be that the change of the enzyme activity affects the expression of EGFR/PI3 K signaling pathway,thereby regulating the proliferation and apoptosis of cells.3 、 There may be a dynamic balance between CD73 hydrolysis enzyme activity and its expression in GC cells.4、CD73 is highly expressed in GC,and its overexpression is correlation with poor prognosis of GC patients.High expression of CD73 is an independent risk factor for the prognosis of GC patients.[Innovation points]CD73 is closely related to the development of tumor and play an important role in the process of tumor proliferation,apoptosis,immune escape,etc.This study applied molecular biology experiment method and combined with the data mining from TCGA database to research the effect of CD73 expression and its hydrolysis enzyme activity on GC BGC-823 cell proliferation,respectively.This study preliminary elaborate the inner relation between CD73 hydrolysis enzyme activity and its expression,and the clinical significance of CD73 expression inGC.The current research provide the experiment basis for CD73 research in gastric cancer.