| Background and Objective:Nasopharyngeal carcinoma originates from the epithelial lining of the nasopharynx and is a highly aggressive head and neck cancer.The 5-year survival rate of nasopharyngeal carcinoma patients has significantly raised due to advances in medical treatment techniques.However,since most nasopharyngeal carcinoma patients are not diagnosed until stage Ⅲ or Ⅳ,30%of nasopharyngeal carcinoma patients will finally have distant metastasis and/or recurrence.Therefore,identifying novel therapeutic targets and predictive biomarkers is important to ameliorate clinical outcomes for nasopharyngeal carcinoma patients.The P2X7 receptor(P2X7R)is a member of the purine receptor family.It is an adenosine triphosphate(ATP)gated ion channel that has been found to be overexpressed in various kinds of cancers.Increased activity of P2X7R is also associated with increased proliferation and survival of various cancer cell types.However,the expression in nasopharyngeal carcinoma(NPC)and its exact role and mechanism remains unclear.The current study aims to explore the expression and effects of P2X7R in nasopharyngeal carcinoma,and to further explore the specific mechanism in nasopharyngeal carcinoma.Method:1.RT-qPCR and Western-blotting were used to detect the m RNA expression level and protein relative expression level in the tumor tissues of NPC patients and normal nasopharyngeal epithelial tissues,the expression level of P2X7R in epithelial cell lines and common nasopharyngeal carcinoma cell lines was detected respectively by RT-qPCR.2.Nasopharyngeal cancer cells 6-10B and CNE-2Z were treated with ATP,Bz ATP,and A438079 or AZD9056 respectively,the expression of P2X7R was detected using Western-Blotting analysis,the expression of P2X7R was knocked down using small interference fragments,and the expression of P2X7R was detected via Western-blotting experiments.Differences in nasopharyngeal carcinoma cell proliferation between treatment groups and control groups were investigated.3.ATP,A438079 or AZD9056,and small interference fragments were used on6-10B and CNE-2Z to knock down the expression of P2X7R.The expression of p-PI3K and p-AKT in the nasopharyngeal carcinoma cells was then detected via Western-blotting.Investigating the difference between treatment group and control group on the proliferation of nasopharyngeal carcinoma cells.4.Nasopharyngeal carcinoma cells were treated with AKT pathway-specific inhibitor(LY294002),and the protein expression of P2X7 receptor and proliferation of nasopharyngeal carcinoma cells were detected.NPC cells were treated with PI3K inhibitor LY294002,and the expression of P2X7R protein was detected,and the effect of LY294002 intervention on the proliferation of NPC cells was detected.5.Establishment of ectopic tumorigenic animal model in nude mice:100μl CNE-2Z cells(about 6×10~6)were injected into the left axillar of nude mice to establish subcutaneous tumor bearing model of nude mice.Different groups of mice were injected respectively with PBS,ATP and AZD9056 periodically to detect the effect of P2X7R activation and inhibition on tumor growth.The nude mice were then kept to observe the tumor size and growth.Results:1.Immunohistochemical staining showed that the expression level of P2X7R in nasopharyngeal carcinoma tissues was significantly higher than that in paired normal nasopharyngeal epithelial tissues.The results of RT-qPCR and Western-blotting experiments showed that the expression level of P2X7R in NPC tissues was significantly higher than that in normal nasopharyngeal epithelial tissues.The expression level of P2X7R in NPC cell lines was also significantly higher than that in normal nasopharyngeal epithelial cells.2.Western-blotting experiments have proven that P2X7R activator improved the expression of P2X7R in nasopharyngeal carcinoma cells,P2X7R antagonists could inhibit the increase of P2X7R expression induced by the activator,and P2X7R knockdown could reduce the expression level of P2X7R in NPC cells.The in vitro experiments showed that P2X7R activators promoted the proliferation of nasopharyngeal carcinoma cells,whereas P2X7R inhibitors inhibited the proliferation of nasopharyngeal carcinoma cells induced by the activators.However,in the absence of ATP,P2X7 receptor antagonists alone had no significant inhibitory effect on cell activity.P2X7R knockdown could reduce the proliferation of nasopharyngeal carcinoma cells.3.Western-blotting experiments proved that P2X7R activator could increase the protein expression levels of P-PI3K and P-AKT,and P2X7R antagonists inhibited the increased protein expression of P-PI3K and P-AKT induced by the activator.P2X7receptor antagonists alone had no significant effect on the protein expression levels of P-PI3K and P-AKT.P2X7R knockdown could reduce the protein expression of P-PI3K and P-AKT in nasopharyngeal carcinoma cells.4.Western-blotting experiments showed that the treatment of PI3K/AKT pathway inhibitor LY294002 inhibited the protein expression levels of P-PI3K and P-AKT.LY294002 combined with A438079 did not show significant synergistic inhibition of P-PI3K and P-AKT protein expression levels.LY294002 inhibited the proliferation of nasopharyngeal carcinoma cells,but no synergistic effect was found when LY294002 was used in combination with A438079.5.The results of the tumor bearing subcutaneous model of nude mice in vivo showed that the ATP treatment significantly induced the growth of tumor in vivo and increased the tumor volume.Treatment of AZD9056 inhibited the ATP-induced tumor growth.Conclusion:1.P2X7R is highly expressed in nasopharyngeal carcinoma,which can promote the proliferation of nasopharyngeal carcinoma;2.P2X7R regulates the proliferation of nasopharyngeal carcinoma cells via activating the PI3K/AKT pathway... |
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