| [Background and Objective]: The negative regulation signal mediated by PD-1/PD-L1 pathway can effectively inhibit the function of T cells and B cells, and plays an important role in the negative regulation of immune response in the body. Recently, it is reported that the expression of PD-L1 is related to the mutation status of EGFR, and the activation of EGFR signaling pathway can increase the expression of PD-L1, but the regulation mechanism is still not clear. IL-6/JAK/STAT3 is an important signaling pathway downstream of EGFR, which plays an important role in promoting the proliferation of tumor cells. At the same time, it was reported that STAT3 could bind to the promoter region of PD-L1, subsequently, promote transcription of PD-L1. The purpose of this paper is to explore if IL-6/JAK/STAT3 signaling pathway is involved in the regulation of PD-L1 by the activation of EGFR and the significance of regulation of cell proliferation by PD-L1.[Methods]:(1) We chosen the lung adenocarcinoma cell lines HCC827, PC-9 and H1975 for our research. Their EGFR mutations were identified by gene sequencing. And we stimulate these cell lines with Gefitinib for 48 h, and then test their IC50 by CCK-8 assay.(2) We chosen the EGFR mutant cell lines(HCC827, PC-9 and H1975) andthe wild type EGFR cell lines(H1299, SPC-A1 and A549) and their expression of PD-L1 were assessed by RT-PCR and western blot.(3) Using different concentrations of EGF stimulated HCC827 and PC-9 cell lines for 48 h,and the expression of PD-L1 were assessed by RT-PCR. Select the concentration of most obvious effect to stimulate cell lines again and use western blot to detect the expression of p-EGFR, PD-L1, p-STAT3 and STAT3.(4) Stimulate HCC827 and PC-9 with different concentrations of Gefitinib, and PD-L1, p-STAT, STAT3 were examined by western blot.(5)Stimulate the HCC827 and PC-9 cell lines with Gefitinib and DMSO respectively and collectthe supernatant at different time, then the amount of IL-6 were assessed by Elisa.(6)Cells were incubated with the specific inhibitor of JAK inhibitor AG490 and the expression of STAT3 was inhibited by siRNA, then the expression of PD-L1 was detected by RT-PCR and western blot. The expression of p-STAT3 and STAT3 were examined by western blot.(7) The expression of PD-L1 were inhibited by siRNA, we detected the cell proliferation of HCC827 and PC-9 with CCK-8 assay and colony formation assay, and the cell apoptosis with a flow cytometer.[Results]:(1) HCC827 and PC9 harbor a deletion in exon 19 of EGFR, while H1975 harbors an activating L858 R mutation in exon 21 as well as a secondary mutation(T790M) in exon 20. HCC827 and PC-9 were sensitive to Gefitinib, while H1975 was resistant to Gefitinib.(2) The PD-L1 expression was significantly higher for cell lines with EGFR mutations than for those with wild-type EGFR.(3)EGFR signaling pathway was activation by the stimulation with EGF,and expression of PD-L1 and p-STAT3 were up-regulated while STAT3 had no obvious change.(4) EGFR signaling pathway was inhibited by Gefitinib, and expression of PD-L1 and p-STAT3 weredecreased while STAT3 had no obvious change.(5) The rise tendency of IL-6 concentration in Gefitinib group was slower than that in the DMSO group.(6) The AG490 could down-regulate the expression of PD-L1 and p-STAT3. After the interference of STAT3 with siRNA, the expression of PD-L1 was decreased.(7) After the interference of PD-L1, the cell proliferation decreased and the apoptosis of cells were increased.[Conclusion]:EGFR-TKI could regulate the expression of IL-6, which could activate JAK/STAT3 signaling pathway, and then regulate the expression of STAT3, ultimately affect the expression of PD-L1 and cell function. The EGFR-TKI could not only inhibit the growth of tumor cell directly but also maintain the immune balance via down-regulating the expression of PD-L1.It provides experimental basis and theoretical basis for the combination of targeted therapy and immune therapy for lung cancer, and its clinical significance remains to be further explored. |
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