A Study Of The Role And Mechanism Of P2X7 Receptor In The Progression Of Colitis-associated Cancer(CAC)

?Objective?P2X7,a ATP-gated cation channel,is a homotrimer composed of three identical subunits,each having two transmembrane domains,a large extra-cellular loop,a short intracellular N-terminal,and a long intracellular C-terminal[1].P2X7 is preferentially expressed on epithelial cells and immune cells like macrophages,dendrite cells,monocyte cells,lymphocytes[1-3] and it has been reported that via P2X7,ATP induces a variety of bioactivities of immune cells,e.g.,enhancing phagocytosis,inducing inflammasome formation,and promoting proinflammatory cytokine release[4,5].The P2X7 in IBD animals obviously promote intestinal inflammation [6,7].In recent years,it is evident that P2X7 is present in many kinds of tumor tissues[8],and ATP is rich in the tumor microenvironment,which can activate P2X7 receptor[9,10].Researches show that P2X7 have an important effect on the growth of many kinds of tumors,even on the process of tumor invasion and metastasis.CAC is closely related to IBD,and it is a typical tumor associated with inflammation.CAC is the tumor originated from intestinal epithelial cells,and it is also confirmed that P2X7 receptor is present in colon cancer cells[8].So as for CAC,P2X7 signaling can affect tumor progression indirectly by regulating inflammatory reaction,or directly by regulating the growth of tumor cells.The purpose of this paper is to study the role and mechanisms of P2X7 receptor in the progression of CAC.?Methods?1.Colitis-associated Cancer InductionAfter female C57BL/6N mice of 6-8 weeks old are injected intraperitoneally(IP)with12.5 mg/kg of AOM working solution,2.5% of DSS drinking water is provided during the second,fifth and eighth week and normal drinking water in the rest of the time.The modeling lasts 10 weeks totally.2.Western BlotLoad 10 ?l protein sample onto SDS-PAGE gel.Electrotransfer to nitrocellulose membrane.Incubate membrane in 1× TBST with 5% w/v nonfat dry milk for 1 hr at room temperature.Incubate membrane and primary antibody(at the appropriate dilution and buffer as recommended in the product datasheet)in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.Wash three times for 5 min each with 15 ml of TBST.Incubate membrane with the species appropriate HRP-conjugated secondary antibody(1:2000)in 10 ml of 1× TBST with 5% w/v nonfat dry milk with gentle agitation for 1 hr at room temperature.Wash three times for 5 min each with 15 ml of TBST.Incubate membrane with mixture of volumetric reagent A and reagent B of Luminol Developer with gentle agitation for 1 min at room temperature.Drain membrane of excess developing solution(do not let dry),wrap in plastic wrap and expose to x-ray film.3.Cell lines and cell cultureIn this experiment,HT29 cells were purchased from Shanghai Yuan Biotechnology Co.ltd..The cell culture medium is Mc Coy's 5A complete medium.Cells were cultured in5% CO2 at 37°C.4.Cell immunofluorescenceSolution: 2g sucrose dissolved in 50 ml 4% paraformaldehyde(PFA),which is used to fix cells.Wash three times for 3 min each with PBS.0.01 mol/L Citrate buffer of p H6.0 is added.Place the culture dish in a water bath at 80°C for 20 min.Cool naturally at room temperature.Wash three times for 3 min each with PBS.Incubate cells and primary antibody overnight at 4°C.Wash three times for 3 min each with PBS.Incubate cells with the species appropriate fluorescence-conjugated secondary antibody in the dark for 1 hr at room temperature.Wash three times for 3 min each with PBS.Adding Hoechst 33258 dye staining,incubate for 10 min at room temperature.Wash three times for 3 min each with PBS.Preserved with glycerin covering on the cells.Photo under fluorescence microscope.5.Cell viability assayIncubate cells and CCK solution for 2 hr in 5% CO2 at 37°C.Read absorbance at 450 nm.6.Cell proliferation assayAdd prepared 10× BrdU solution to plate wells,for a final 1× concentration.Place cells in incubator for 4 hr.Remove medium.Add 100 ?l/well of Fixing/Denaturing solution.Keep the plate at room temperature for 30 min.Remove solution.Add 100?l/well of prepared 1× detection antibody solution.Keep the plate at room temperature for1 hr.Remove solution and wash plate 3 times with 1× Wash Buffer.Add 100 ?l/well of prepared 1× HRP-conjugated secondary antibody solution.Keep the plate at room temperature for 30 min.Remove solution and wash plate 3 times with 1× Wash Buffer.Add 100 ?l TMB Substrate.Incubate for 30 min at room temperature.Add 100 ?l STOPSolution.Read absorbance at 450 nm.7.Detection of apoptosisWash cells twice with cold PBS and then resuspend cells in 1× Binding Buffer at a concentration of 1*106 cells/ml.Transfer 200 ?l of the solution(1*105 cells)to a 5 ml culture tube.Add 5 ?l of FITC Annexin V.Gently cortex the cells and incubate for 15 min at room temperature in the dark.Add 5 ?l of PI.Gently cortex the cells and incubate for 4min at room temperature in the dark.Analyze apoptosis by flow cytometry with 1 hr.8.Cell cycle detectionWash cells twice with cold PBS and then add 5 ml of 75% ethanol.Gently cortex the cells and place the cells overnight at 4 °C.Wash cells twice with cold PBS.Add PI/Rnase Staining Buffer and gently cortex the cells.Incubate for 40 min at room temperature in the dark.Analyze cell cycle by flow cytometry with 1hr.?Results?1.The selective P2X7 antagonist BBG inhibited the recovery of body weight and decreased the survial of CAC mice,and promoted tumorigenesis of CAC mice and phosphorylation of STAT3 from the polyps colon tissue sections of CAC mice.2.P2X7 agonist Bz ATP impeded tumorigenesis of CAC mice and phosphorylation of STAT3 from the polyps colon tissue sections of CAC mice.3.P2X7 receptor was present in HT29 cell.4.P2X7 agonist BzATP induced vacuolization of HT29 cell.5.P2X7 agonist BzATP induced HT29 to arrest at G1 cell cycle,inhibited the proliferation of HT29 cells and HT29 cell activity,while had no significant effect on cell apoptosis.6.P2X7 agonist Bz ATP inhibited the phosphorylation of STAT3 in HT29 cells.?Conclusions?1.P2X7 signaling plays a role in blocking the progression of CAC in mice.2.Activation of P2X7 receptor in human intestinal tumor cell can directly inhibit its proliferation,which is maybe mediated by inhibiting phosphorylation of the STAT3 in the cell.3.P2X7 activation can inhibit phosphorylation of STAT3 from the polyps colon tissue sections of CAC mice,which may be one of the underlying mechanism of P2X7 signaling inhibiting the progression of CAC.