Association Between PD-L1 And Radiotherapy Sensitivity In Esophageal Squamous Cell Carcinoma

Objective:1.The purpose of this research to investigate the relationship of PD-L1 with clinicopathological characteristics,radiotherapy response and prognosis of patients with ESCC.2.The purpose of this experiment to study the time and dose-effect in expression of PD-L1 gene after irradiation and observe the influence extend of PD-L1 siRNA to the cell proliferation,cell cycle,apoptosis and sensitivity of radiotherapy of Eca109 cell of human esophageal carcinoma,in order to explore the mechanism about sensitivity of radiotherapy.Methods:1.Ninety esophageal squamous cell carcinoma tissue samples and 20 normal esophageal mucosa samples were collected and their paraffin-embedded sections were immunohistochemically stained to indicate the expression levels of PD-L1.Staining intensity and proportion of the positive cells were calculated and statistically analyzed to explore the relationship between the PD-L1 expression and the radiotherapy effect,prognosis and the clinical and pathological characteristics,including the tumor size,lymphatic metastasis,TNM grade and pathological grade.2.The level of mRNA and protein expression of PD-L1 in Eca109 cells after different dose of X-ray irradiation were determined using reverse transcriptase-polymerase chain reaction(RT-PCR)and western blotting(WB),respectively.The cell proliferation ability was measured by CCK8 assay method.The purpose of the test to study the time and dose-effect and discuss the best dose of the follow-up research.3.The level of mRNA and protein expression of PD-L1 in Eca109 cells before and after interference were determined using reverse transcriptase-polymerase chain reaction(RT-PCR).CCK-8 and FCM respectively to detect the cell proliferation and apoptosis.Compare the difference of proliferation,cell cycle,apoptosis and sensitivity of radiotherapy of Eca109 cell that radiation of 6Gy was done with PD-L1 siRNA transfected Eca109 cells,control siRNA transfected Eca109 cells(negative control)and blank Eca109 cells(blank control).In order to preliminary explore the mechanism of the sensitivity of radiotherapy.Results:1.Association between the expression of PD-L1 protein and ESCC1.1 PD-L1 expression in ESCCThe results of immunohistochemically stained showed that,in 90 cases of esophageal carcinoma tissues,the positive rate was 56.7 %(51/90),significantly higher than the normal esophageal mucosa tissues 15%(3/20).1.2 Association between the expression of PD-L1 and and clinicopathologic feature in ESCCThe expression of PD-L1 protein was not correlated with age,gender,the site of lesion,the maximum diameter of tumor,the length of tumor,the local aggressive of tumor,the primary tumor stage,clinical stage and primary tumor volume(P > 0.05).PD-L1 expression levels was statistically correlated with the lymphatic metastasis(P<0.05).1.3 The relationship between PD-L1 expression and radiosensitivity90 cases of esophageal patients were given efficacy evaluation by esophagus barium meal X-ray examination in three months after radiotherapy,in which 68 cases of radiation sensitivity(CR+PR);NR(21 cases)radiation resistance.Positive PD-L1 expression rate was 63.2%(43/68)in the radiosensitivity group,however 39%(8/22)was negative expression in radioresistance group.Pearson's chi-aquared test showed that there was statistic difference between the groups(?2=4.888,P<0.05)1.4 The relationship between PD-L1 expression and prognosis of the accept radical radiotherapy of esophageal squamous carcinoma.3-year progress free survival rate in positive PD-L1 expression group was 28.4%,one in negative group was 19.8%.3-year overall survival rate in positive PD-L1 expression group was 31.9%,one in negative group was 20.9%.Kaplan-Meier univariate survival analysis showed that two groups has no statistically significant difference in progress free survival rate and overall survival rate.Cox proportional hazard model multivariate analysis also revealed that the PD-L1 could not be considered as a prognosis indicators of ESCC.2.Expression of PD-L1 in Eca109 cells before and after different dose of X-ray irradiation2.1 PD-L1 expression in Eca109 cellsThe results of RT-PCR and WB showed that the level of mRNA and protein expression of PD-L1 in Eca109 cells were significantly higher than the HET-1A cells.2.2 Expression of PD-L1 in Eca109 cells before and after irradiationThere was a significantly increased of the expression quantity of PD-L1 after irradiated at different time comparing with those without irradiated cells.The expression level of PD-L1 reached peak at 12 hours by 3Gy irradiated given.however,the 6Gy and 9Gy reached a peak in 24 hours.With extended after irradiation time,expression of PD-L1 gradually decreased.2.3 Proliferation ability of Eca109 cells before and after irradiationThere was a significantly decreased of the cell proliferation ability after different dose of X-ray irradiation at different time comparing with those without irradiated cells,especially at 24 h There was a statistically significant fall in cell proliferation after 24 hours postirradiation.3.The influence of Eca109 cells after slience PD-L1 by RNAi technology3.1 PD-L1 expression in Eca109 cells after transfectionContrasted with Eca109 and Eca109-N group,the expression of PD-L1 mRNA decreased 72.5%(1.02±0.03)and 71.7%(0.99±0.03),respectively.The expression of PD-L1 of Eca109-N group dropped 5% compared with Eca109 group but they did not achieve statistical significance.3.2 After transfection,PD-L1 effect on Eca109 cell proliferation levels before and after irradiationIt had no difference between the cell proliferation of every group in 36 h.Contrasted with Eca109 and Eca109-N group,the cell proliferation rates of Eca109-P were gradually decreased at 48 h.The cell proliferation of every group decreased after 24 hours postirradiation of 6Gy.With extended after irradiation time,cell proliferation ability gradually increased.Contrasted with Eca109 and Eca109-N group,the cell proliferation rates of Eca109-P were Dramatically decreased at 36 h and 48 h by 6Gy irradiated given.3.3 Down-regulated the expression of PD-L1,effect on esophageal cancer cell cycle before and after irradiation.The distribution of cell cycle in G0/G1,G2/M and S stage were not different with none irradiated in Eca109,Eca109-N and Eca-109-P.compare with other group,G2/M stage of Eca109-P group rose,dropped in S stage.but retardance of G2/M was observed by irradiated 6Gy,and stage G2/M were significantly lowered than Eca109 and Eca109-N.Percentage of stage G0/G1 in Eca109-P cells line with irradiated was higher than Eca109 and Eca109-N by irradiated 6Gy.There was not influenced of S stage cell by irradiated.3.4 After transfection,PD-L1 effect on esophageal cancer cell apoptosis rates before and after irradiationCell apoptosis rate significantly increased by transfected than those in Eca109 with no transfected.Contrasted with Eca109 and Eca109-N group,Eca109-P group owned the higher cell apoptosis rates.Conclusions:1.The level of PD-L1 protein expression is increased in ESCC tissues compared to the normal esophageal mucosa tissues of normal people.PD-L1 may be a novel predictive marker for response to the radiotherapy and metastatic potential in patients with ESCC.But positive PD-L1 cannot as one of prognosis predictors in ESCC patients accomplished curative radiotherapy.2.The level of PD-L1 protein expression is increased in ESCC tissues compared to the normal esophageal mucosa tissues of normal people.Protein expression of PD-L1 is increased along with the increase of radiation dose and dependently relate to dose and time.3.Cell proliferation activity is inhibited,and induced the retardance of stage G0/G1,and increased the cell apoptosis of Eca109 cells when PD-L1 gene expression down regulation by siRNA method.This experiment suggests PD-L1 decline proliferation ability of Eca109 cells,induce the G0/G1 phase to the G2/M phase transformation,increase the proportion of proliferating Cells,thereby increase the radiosensitivity of Eca109 cells.