Study On Multiple Signaling Pathways In Esophageal Squamous Cell Carcinomas Involved In The Regulation Of PD-L1 Gene Expression

Objective: To study whether Survivin is involved in TNF-α induced increase of pd-l1 expression in esophageal squamous cell carcinoma(Eca109)cells,and preliminarily identify the signaling pathway related to pd-l1 expression.Methods: 1)Tumor necrosis factor alpha(TNF-α)was used to induce esophageal squamous cell carcinoma Eca109 to increase the expression of PD-L1.2)The major intracellular signaling pathways in which TNF-and pd-l1 expression is induced were screened for 1.5,3,6,12,24 hours by the intervention of ERK,MEK,JNK,PI3K/AKT,STAT3,PKA,PKA,PKC,Hedgehog,CaMK2 signaling pathway inhibitors SCH772984,PD98059,SP600125,LY294002,AG490,H89,Staurosporine,Vismodegib,KN62 in Eca109 cells.3)Use Flag-Survivin overexpression vector,Survivin inhibitor YM155 and the main signaling pathway inhibitors selected to intervene in Eca109 cells for 3,12,and 24 hours.4)Supernatant of cell culture medium was collected from different treatment groups and exosomes were extracted by ultra-high speed centrifugation.In this study,Western blot protein electrophoresis was used to detect the changes in the expression of ERK,MEK,JNK,PI3K/AKT,p-stat3,PKA,PKC,Hedgehog,CaMK2,pd-l1 and Survivin at the protein levels of the related signaling pathways after the intervention of inhibitors.Flow cytometry was used to detect the changes of apoptosis.Exosomes from different treatment groups were extracted by ultra-high speed gradient centrifugation and identified by electron microscopy and Western blot.Results: 1)After 1.5,3,6,12 and 24 hours of induction of Eca109 cells by TNF-α,Western blot results showed that the expression of pd-l1 protein showed an increased trend,confirming that the expression of pd-l1 in Eca109 cells was increased by TNF-α induction.2)Among the nine signaling pathways of ERK,MEK,JNK,PI3K/AKT,STAT3,PKA,PKC,Hedgehog and CaMK2,only the inhibitor AG490 of the STAT3 signaling pathway was used,and the expression levels of p-stat3,Survivin and pd-l1 were all down-regulated,and there was a synergistic effect between STAT3,pd-l1 and Survivin.3)In Eca109 cells transfected with flag-survivin vector,the STAT3/ pd-l1 signaling pathway was up-regulated with the increase of Survivin expression.Both Survivin inhibitor YM155 and STAT3 inhibitor AG490 inhibited the expression of Survivin,and used both the inhibitory effect of the inhibitor is more obvious.4)AG490 inhibited the expression of STAT3 and pd-l1,and increased the rate of late apoptosis of tumor cells.5)The amount of Survivin and pd-l1 in exosomes extracted from the cell culture medium was small,and there was no difference between each group.Conclusion: STAT3 is the main intracellular signal transduction pathway of TNF-α induced high expression of PD-L1 in esophageal squamous cell carcinoma Eca109 cells.Survivin and STAT3 have synergistic effect in the regulation of pdl1 expression,and there is STAT3/Survivin/ pd-l1 pathway in Eca109 cells.