Prostaglandin E2 Increases The Expression Of SOX2 In Residual Nasopharyngeal Carcinoma Cells After Radiation Through EP2

Radiotherapy?RT?has become an indispensable method for treatment of nasopharyngeal carcinoma?NPC?,but local recurrence and distant metastasis are two important reasons for treatment failure.More surprisingly,RT itself may be the trigger for these malignant processes..Cancer stem cells?CSCs?have the ability of self-renewal,survive treatment,continue to proliferate and differentiate to initiate malignant process,leading to treatment failure.The ionizing radiation?IR?has been proved to induce the phenotype of stem cells and promote the malignant process of tumors in many cancers.Previous studies by our research group also showed that IR can improve the expression of NPC residual cancer cell stem cell phenotype.IR-induced biological factors can induce pro-inflammatory immune response in tumor microenvironment.Prostaglandin E2?PGE2?is the most abundant prostaglandin in various human malignant tumors,which plays an important role in promoting tumor growth.Moreover,our research group has previously confirmed that IR can induce PGE2 secretion in irradiated NPC.We simulated the clinical RT segmentation of NPC,and used 2Gy⁶⁰Co?rays to irradiate NPC cells for several times to establish residual NPC cell lines after radiation.With the aid of cell counting kit-8?CCK8?experimental to screen the half maximal inhibitory concentration?IC50?of celecoxib on residual NPC cells.The effect of celecoxib on the stem cell phenotype of residual NPC cells was verified by in vitro suspension spherulation,qRT-PCR and WB experiments.After blocking endogenous PGE2,qRT-PCR and WB were used to detect the effect of exogenous PGE2 on phenotypic markers of NPC residual cancer cells.Finally,residual NPC cells with prostaglandin E2 receptor?EP?2/4 were established by lentivirus vector infection.Using it as a research model,WB and cellular immunofluorescence assay were used to detect the differences in the expression of stem cell phenotype markers in residual NPC cells that stabilized the Modulation of EP2/EP4 only under the action of exogenous PGE2.From the perspective of tumor inflammatory microenvironment after radiation,this study explored the effect and mechanism of exogenous PGE2 on stem cell phenotype in residual NPC cells through its receptor,hoping to find neoadjuvant therapy and new targeted therapy direction for NPC after RT,reduce the occurrence of malignant process,and thus improve the therapeutic effect.Aims:1.To clarify the effect of celecoxib on the stem cell phenotype of residual NPC cells.2.To confirm the regulatory of exogenous PGE2 on stem cell phenotype markers in residual NPC cells based on the inhibition of endogenous PGE2 by celecoxib.3.To explore the molecular pathway through which exogenous PGE2 increases the expression of stem cell phenotype markers of in residual NPC cells.Methods:1.CNE-1 and 5-8F cells were irradiated with 2Gy⁶⁰Co?ray for 7 consecutive times to establish the residual cancer cell line model after radiation.2.Celecoxib was used to screen the IC50 of residual CNE-1 and 5-8F cells using CCK8 assay.3.Effects of celecoxib on stem cell phenotype markers SOX2,OCT4 and KLF4were detected by vitro suspension tumorsphere formation assays,qRT-PCR and WB in residual CNE-1 and 5-8F cells.4.On the basis of inhibiting endogenous PGE2 in residual CNE-1 and 5-8F cells by cexoxib,exogenous PGE2 was added to stimulate NPC residual cancer cells,and the effects of exogenous PGE2 on stem cell phenotype markers SOX2,OCT4 and KLF4 were detected by qRT-PCR and WB experiments.5.Residual CNE-1 and 5-8F cells were infected with lentivirus vectors,and stable down-regulated EP2/EP4 cells were established respectively.6.Residual CNE-1 and 5-8F cells with stabilized regulation of EP2/EP4 were used as research models.SOX2 expression in residual CNE-1 and 5-8F cells of EP2/EP4 was detected by WB and cell immunofluorescence assay under the condition of exogenous PGE2 after blocking endogenous PGE2 by celeoxib.Results:1.Residual CNE-1 cell lines E-R7 were obtained by irradiation CNE-1 cells for 7consecutive times;the same treatment was applied to 5-8F cells to obtain the residual 5-8F cell lines F-R7.2.The IC50 of residual CNE-1 and 5-8F cells were 131?M and 98?M respectively.3.The results of in vitro suspension tumorsphere formation assays,qRT-PCR and WB experiments showed that celecoxib could reduce the tumorsphere formation ability and the expression of stem cell phenotype markers SOX2,OCT4 and KLF4 in residual CNE-1 and 5-8F cells.4.qRT-PCR and WB results showed that,on the basis of inhibiting endogenous PGE2,exogenous PGE2 could increase the expression of SOX2,but could not improve the expression of OCT4 and KLF4 in residual CNE-1 and 5-8F cells.5.WB and immunofluorescence results showed that,based on the inhibition of endogenous PGE2 by celecoxib,down-regulated EP2 could inhibit the promotion of SOX2 in residual CNE-1 and 5-8F cells by exogenous PGE2.Conclusions:1.Celecoxib can inhibit the stem cell phenotype in residual CNE-1 and 5-8F cells.2.After celecoxib inhibited endogenous PGE2 production,exogenous PGE2increased SOX2 expression,but not OCT4 and KLF4 expression in residual CNE-1 and5-8F cells.3.After celecoxib inhibits the production of endogenous PGE2,exogenous PGE2increases the expression of SOX2 through EP2 but not EP4 in residual CNE-1 and 5-8F cells.