| Objective:1.To test the expression of interleukin 6(IL-6),signal transducer and activator of transcription 3(STAT3)and programmed death ligand 1(PD-L1)in Esophageal Squamous Cell Carcinoma,and to explore the relationship of PD-L1,IL-6 and STAT3 with clinicopathological characteristics and prognosis of esophageal squamous cell carcinoma..2.There is a synergistic effect between radiotherapy and immunotherapy in the treatment of esophageal cancer,but the mechanism by which radiotherapy regulates PD-L1 expression of esophageal cancer cells is not clear.The IL-6/STAT3 signaling pathway may be involved.To study differences in PD-L1,IL-6 and STAT3 protein expressions in the esophageal carcinoma cell lines Eca109,KYSE150 and TE1 after ionizing radiation and the effects of radiotherapy on cell proliferation,cell invasion and the cell cycle.Methods:1.RT-PCR was used to detect PD-L1,IL-6,and STAT3 mRNA expression in cancerous and adjacent normal tissues.Western blot and immunohistochemical detection of PD-L1,IL-6,and STAT3 protein expression was performed in adjacent normal and cancerous tissues in 40 patients.The correlation of PD-L1,IL-6 and STAT3 protein expression with tumor invasion depth,tumor size,pathological grade of lymph node metastasis,other clinicopathological characteristics and prognosis of esophageal squamous cell carcinoma.2.After applying different doses of X-ray irradiation to the esophageal cancer cell lines(Eca109,KYSE150,and TE1),RT-PCR and Western blotting were used to detect PD-L1 mRNA and protein expression levels and STAT3 and P-STAT3 protein expression levels.Enzyme-linked immunosorbent assays(ELISAs)were performed to examine the effects of different doses of radiotherapy on IL-6 protein expression levels in Eca109,KYSE150,and TE1 cells,and detect the expression of PD-L1 in cell lines after IL-6/STAT3 blocker(AG490)combined with X-ray irradiation.Cell Counting Kit-8(CCK-8)assays were used to test the effects of different doses of radiotherapy on the proliferation of Eca109,KYSE150,and TE1 cells.In addition,the time-dose effect relationship of PD-L1 was studied to explore the optimal radiation dose for subsequent experiments.At the optimal radiation dose,Transwell assays were used to detect changes in the invasion and proliferation of cell clones of the three cell lines after radiotherapy,and cell cycle changes in the three cell lines were detected by flow cytometry.Results:1.The results of esophageal cancer tissues.1.1 The mRNA levels of PD-L1,IL-6,and STAT3 in 70 esophageal squamous cell carcinoma patients were significantly different between cancerous and adjacent normal tissues(P<0.05).1.2 Compared with 40 esophageal squamous cell carcinoma tissues,28 esophageal squamous cell carcinoma tissues showed significantly higher PD-L1 expression(28/12)(P<0.01);29 esophageal squamous cell carcinoma tissues showed significantly higher STAT3 expression(29/11)(P<0.05);and 27 esophageal squamous cell carcinoma tissues showed significantly higher IL-6 expression(27/13)(P<0.05).1.3 PD-L1 expression showed a positive correlation with T stage(?2=6.791,P<0.05),lymph node metastasis(?2=4.405,P<0.05),and clinical stage(?2=4.571,P<0.05).STAT3 expression showed a positive correlation with T stage(?2=5.021,P<.05).IL-6 expression showed a positive correlation with T stage(?2=8.780,P<0.05)and tumor site(?2=6.420,P<0.05).In addition,PD-L1 expression showed a positive correlation with IL-6(?2=7.033,P<0.05)and STAT3(x2=6.114,P<0.05),and STAT3 showed a positive correlation with IL-6(?2=4.890,P<0.05).1.4 The relationship between PD-L1,IL-6,STAT3 expression and prognosis of the accept radical surgery of esophageal squamous carcinoma.The 1-year disease-free survival rate in the PD-L1 high expression group was 71.4%,and the low expression group was 91.7%;the 1-year disease-free survival rate in the IL-6 high expression group was 70.4%,and the low expression group was 92.3%;the STAT3 high expression group was 1 year free of disease The survival time was 72.4%,and the low expression group was 90.9%.The Kaplan-Meier univariate analysis of PD-L1,IL-6,and STAT3 showed no significant difference in disease-free survival(DFS)between high and low expression(P>0.05).Cox proportional hazards model multivariate analysis found that the largest tumor diameter and clinical stage were independent prognostic factors for esophageal squamous cell carcinoma,while the expression levels of PD-L1,IL-6,and STAT3 were not independent prognostic factors for esophageal squamous cell carcinoma.2.The results of esophageal carcinoma cell lines.2.1 The mRNA expression level and the protein expression levels of PD-L1 was significantly higher in the esophageal cancer cell lines KYSE150,TE1 and Eca109 than in the normal esophageal cell line HET-1A(P<0.05).2.2 After 3,6,9 and 12 Gy irradiation,the PD-L1 mRNA levels first increased and then decreased in the three esophageal cancer cell lines(Eca109,KYSE150,and TE1).PD-L1 expression peaked in the three cell lines 12 hours after irradiation and was significantly higher in irradiated cells than in nonirradiated cells(P<0.05).Moreover,at an extended time after irradiation,PD-L1 expression was decreased(P<0.05).2.3 PD-L1 protein expression in the esophageal cancer cell lines(Eca109,KYSE150 and TE1)first increased and then decreased after each dose of irradiation.PD-L1 protein expression in the three esophageal cancer cell lines peaked 48 hours after 6 Gy irradiation(P<0.05).At an extended time after irradiation,PD-L1 protein expression gradually decreased(P<0.05).2.4 P-STAT3 protein expression in the esophageal cancer cell lines(Eca 109,KYSE150,and TE1)first increased and then decreased after each dose of irradiation,and Eca109 protein expression peaked 48 hours after irradiation with 6 Gy(P<0.05).At an extended time after irradiation,P-STAT3 protein expression decreased(P<0.05),and P-STAT3 protein expression peaked 24 hours after irradiation with 6 Gy in KYSE150 and TE1 cells(P<0.05).At an extended time after irradiation,the expression level of P-STAT3 decreased gradually(P<0.05).2.5 After irradiation with different doses of radiation,the expression of IL-6 protein in esophageal cancer cell lines KYSE150,TE1,Eca109 increased after each dose of irradiation,and the increase rate reached the peak at 12h after irradiation(P<0.05),with the time after irradiation Prolonged,the rate of increase in IL-6 protein expression gradually decreased(P<0.05).2.6 Compared with nonirradiated cells,the proliferation levels of Eca109,KYSE150 and TE1 esophageal cancer cells in the different dose groups decreased at different time points after irradiation,and the inhibitory force increased with the increase in dose and time after radiotherapy(P<0.05).2.7 When the IL-6/STAT3 signaling pathway was blocked by the specific inhibitor AG490,Western blot showed that radiotherapy could not increase the expression of PD-L1.2.8 Compared with nonirradiated cells,the clone formation ability of the three cell lines(Eca109,KYSE150 and TE1)was inhibited to varying degrees 14 days after irradiation with 6 Gy(P<0.05).2.9 Compared with nonirradiated cells,the invasion ability of the three cell lines(Eca 109,KYSE150 and TE1)was significantly affected 48 hours after irradiation with 6 Gy,and the invasion ability of the three esophageal cancer cell lines was lower than that of the nonirradiated cell lines(P<0.05).2.10The three cell lines(Eca109,KYSE150 and TE1)were exposed to 6 Gy irradiation for 48 hours,and the proportion of cells in G2/M phase was higher in exposed cells than in nonexposed cells(P<0.05),while the proportion of cells in S phase did not change significantly(P>0.05).Conclusion:1.PD-L1,IL-6 and STAT3 are highly expressed in tumor tissues,and the later the T stage was,the higher the relative expression of PD-L1,IL-6 and STAT3 were.2.The expression of PD-L1,IL-6 and STAT3 cannot be used as predictors of the prognosis of patients with esophageal cancer after radical surgery,and the IL-6/STAT3 signaling pathway protein may be a potential biomarker to predict the effect of immunotherapy by affecting the expression of PD-L1.3.Radiotherapy can regulate the expression of PD-L1 through the IL-6/STAT3 signaling pathway. |
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