| Background:Gastric cancer(GC)is a common malignant tumor in the digestive system,which seriously affects people’s physical and mental health.Although GC is treated with multiple methods,which improves the survival rate and quality of life of patients,but the pathogenesis of GC is complex and involves many factors.Recurrence and metastasis are the main causes of death of patients.Therefore,exploring and excavating new molecular basis in the pathogenesis of GC and improving the treatment of GC are problems that need to be solved urgently.Studies have confirmed that the ATP ion channel P2X7 purinergic receptor has an important regulatory effect on the growth,apoptosis,invasion and metastasis of tumor cells.However,whether P2X7 purinergic receptor has a regulatory effect on the growth and metastasis of GC is still lacking.Therefore,the purpose of this study is to explore the effects of P2X7 purinergic receptor on the growth and metastasis of GC and related molecular mechanisms via in vivo and in vitro experiments,so as to provide a new theoretical basis and data support for the treatment of GC.Objection:To clarify the relationship between P2X7 receptor and the clinicopathological characteristics of patients with GC,to further explore the role of P2X7 receptor on the proliferation,migration and metastasis of GC and the related molecular mechanisms,so as to provide a certain reference value for the prevention and treatment of GC in the future.Methods:1.Clinical sample collectionH-E,immunohistochemistry,immunofluorescence,q RT-PCR and Western-blotting were performed to detect the expression of P2X7 receptor in GC tissues and adjacent tissues.According to the histopathological scoring standard,the high and low expression of P2X7 receptor in GC tissues were scored,and the correlation between P2X7 receptor and clinicopathological characteristics was analyzed.Kaplan-Meier survival curve was used to analyze the correlation between P2X7 receptor and the overall survival prognosis of patients with GC.2.In vitro experiments(1)Western-blotting,q RT-PCR and immunofluorescence were performed to measure the P2X7 receptor expression and location in GC cell lines(AGS and HGC-27)and normal gastric mucosa epithelial cell lines(GES-1).Immunofluorescence was used to measure the fluorescence intensity of ATP and Bz ATP and the use of antagonist A438079 and AZD9056 on P2X7 receptor-labeled cells.(2)To understand the functional characteristics of P2X7 receptor in GC cells:Flu-3AM assay was performed to detect the effect of P2X7 receptor on calcium ions.(3)Cell scratches,Transwell migration and invasion assays were performed to detect the migration and invasion ability of P2X7 receptor on cells.(4)EDU,CCK-8 and plate cloning experiments were performed to detect the effect of P2X7 receptor on cell proliferation activity.(5)PAS glycogen staining assay was used to detect the expression of glycogen in cells.(6)Actin and methyl violet staining assays were performed to detect the changes in cell actin filaments and cell morphology caused by P2X7 receptor activation.(7)Western-blotting was performed to measure the expression of EMT/metastasis related genes(E-cadherin,Vimentin,Snail,N-cadherin,Zeb1,MMP-2 and MMP-9)in GC cells.Immunofluorescence was used to detect the changes in the fluorescence intensity of E-cadherin and Vimentin by P2X7 receptor activation.(8)si RNA was used to transfect GC cells to knock down the expression of P2X7receptor to further verify the changes of P2X7 receptor on the proliferation,migration and invasion of GC cells.(9)Western-blotting was used to detect the expression of P2X7 receptor on p-AKT and p-GSK-3beta.The P13/AKT signaling pathway inhibitor LY294002 was used to treat GC cells.Cell scratches,Transwell invasion,CCK-8 and PAS staining experiments were used to detect the changes in the proliferation,migration and invasion of GC cells.3.In vivo experiments(1)Establishment of an animal model of ectopic tumor formation in nude mice:100μl of AGS cells(approximately 4×10~6 cells)were injected subcutaneously into the outer thigh of nude mice for ectopic tumor formation,and the effect of P2X7 receptor activation on tumor growth was detected(measured the size of the tumor).(2)Tumor histological analysis:H-E,IHC,immunofluorescence and IF-TUNEL were used to detect the changes of P2X7 receptor,VEGF and Ki67 and the changes of apoptotic cells in tumor tissues.Results:1.Compared with the adjacent tissues,H-E pathological section showed that the structure of GC tissues was unclear and the pathological damage was obvious seen.Expression of P2X7 receptor protein and m RNA in GC tissues increased significantly.The expression score of P2X7 receptor in GC tissues was higher than that in adjacent tissues.High expression of P2X7 receptor was significantly correlated with lymph node metastasis,vascular invasion,TNM staging and poor overall survival prognosis,but had no obvious correlation with patient age,gender,invasion depth and location.2.(1)P2X7 receptor was expressed on the membrane of GC cells.ATP and Bz ATP increased the fluorescence intensity of P2X7 receptor-labeled cells,while A438079 and AZD9056 reduced the increase in fluorescence intensity of P2X7receptor-labeled cells induced by ATP.(2)ATP and Bz ATP significantly increased the intracellular calcium influx,while A438079 reduced the ATP-induced calcium influx.(3)Activation of P2X7 receptor promoted the proliferation,migration and invasion of GC cells,while the use of antagonists and knockdown of P2X7 receptor expression by si RNA reversed the above phenomena.In the absence of ATP,the application of P2X7 receptor antagonist alone had no obvious inhibitory effect on the proliferation,migration and invasion of GC cells.(4)P2X7 receptor activation enhanced the stress response of actin filaments and changes in cell morphology,while A438079 or AZD9056 inhibited these changes induced by ATP.(5)Activation of P2X7 receptor increased the intracellular glycogen content,while the use of antagonists and knockdown of P2X7 receptor expression by si RNA reduced the intracellular glycogen content.Moreover,on the basis of si P2X7 treatment,the use of ATP increased glycogen accumulation in GC cells.(6)P2X7 receptor activation increased the expression of EMT/metastasis-related genes(Vimentin,Snail,N-cadherin,Zeb1,MMP-2 and MMP-9),while decreased the expression of E-cadherin.ATP and Bz ATP enhanced the fluorescence intensity of Vimentin and reduced the fluorescence intensity of E-cadherin.(7)In vivo experiment results showed that ATP induced tumor growth in nude mice,while AZD9056 inhibited ATP-induced tumor growth.Moreover,ATP increased the expression of P2X7 receptor,VEGF and Ki67 in tumor tissues,and reduced the apoptosis rate of cells in tumor tissues.Conversely,the application of AZD9056 reversed the above phenomenon.3.Activation of P2X7 receptor increased the expression of p-AKT and p-GSK-3beta,while inhibition of P2X7 receptor activity reduced the expression of p-AKT and p-GSK-3beta.Knockdown of P2X7 receptor expression in cells by si RNA reduced the expression of p-AKT and p-GSK-3beta,and further verified the above results.Furthermore,the use of P13/AKT signaling pathway inhibitor LY294002inhibited the proliferation,migration and invasion of GC cells.Conclusion:High expression of P2X7 receptor is obviously related to the clinicopathological characteristics of patients with GC.Activation of P2X7 receptor promotes the growth,migration and invasion of GC cells.The possible mechanism is through the activation of P13/AKT/GSK-3beta signaling.These data indicate that P2X7 receptor may become a new target for the treatment of GC,and provide a new theoretical basis and data support for the treatment of GC in the future. |
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