摘要
目的 通过构建稳定敲减PRSS3以及在敲减PRSS3的基础上过表达FGFR2的A549细胞模型,探讨PRSS3通过FGFR2对非小细胞肺癌(NSCLC)细胞免疫逃逸的影响及机制.方法 A549细胞分为sh-NC组、sh-PRSS3组、sh-PRSS3+ov-NC组和sh-PRSS3+ov-FGFR2组,分别转染相应慢病毒.Western blot检测A549细胞中PRSS3、FGFR2、p-JAK2、JAK2、p-STAT3、STAT3和PD-L1蛋白表达;CCK-8检测A549细胞增殖能力;Transwell检测A549细胞迁移和侵袭能力;将A549细胞与CD8+T细胞共培养,台盼蓝染色和流式细胞术检测CD8+T细胞的细胞活力和凋亡率;共培养细胞培养液上清中sPD-L1、IFN-γ、TNF-α、颗粒酶B和穿孔素水平采用ELISA进行检测.结果 与sh-NC组相比,sh-PRSS3组和sh-PRSS3+ov-NC组A549细胞中PRSS3、FGFR2、p-JAK2、p-STAT3和PD-L1蛋白表达以及A549细胞的增殖活性、迁移细胞数和侵袭细胞数显著降低,与A549细胞共培养的CD8+T细胞活力以及细胞培养液上清中IFN-γ、TNF-α、颗粒酶B和穿孔素水平显著升高,细胞凋亡率以及细胞培养液上清中sPD-L1水平显著降低;与sh-PRSS3组和sh-PRSS3+ov-NC组相比,sh-PRSS3+ov-FGFR2组A549细胞中PRSS3、FGFR2、p-JAK2、p-STAT3和PD-L1蛋白表达以及A549细胞的增殖活性、迁移细胞数和侵袭细胞数显著升高,与A549细胞共培养的CD8+T细胞活力以及细胞培养液上清中IFN-γ、TNF-α、颗粒酶B和穿孔素水平显著降低,细胞凋亡率以及细胞培养液上清中sPD-L1水平显著升高.结论 PRSS3通过上调FGFR2表达诱导NSCLC细胞免疫逃逸,其机制可能与激活JAK2/STAT3信号通路诱导的PD-L1表达上调有关.
Abstract
Objective To investigate the role of PRSS3/FGFR2 in immune escape of non-small cell lung cancer(NCLC)cells by using an A549 cell model which stably knocks down PRSS3 and overexpresses FGFR2 on the basis of PRSS3.Methods A549 cells were divided into sh-NC group,sh-PRSS3 group,sh-PRSS3+ov-NC group and sh-PRSS3+ov-FGFR2 group,and were transfected with corresponding lentiviruses respectively.Western blot was used to detect the expression of PRSS3,FGFR2,p-JAK2,JAK2,p-STAT3,STAT3 and PD-L1 in A549 cells of different groups;CCK-8 was used to detect the proliferation of theses A549 cells;Transwell was used to detect the migration and invasion ability of theses A549 cells.Furhtermore,CD8+T cells were co-cultured with these A549 cells,and trypan blue staining and flow cytometry were used to detect the viability and apoptosis rate of the CD8+T cells,respectively.The levels of sPD-L1,IFN-γ,TNF-α,granzyme B and perforin in the supernatant of co-cultured cells were detected by ELISA.Results Compared with sh-NC group,sh-PRSS3 group and sh-PRSS3+ov-NC group demonstrated lower expressions of PRSS3,FGFR2,p-JAK2,p-STAT3 and PD-L1 proteins,as well as lower levels of proliferation activity,the number of migrating cells and the number of invading cells of A549 cells.Furthermore,in the supernatant of co-culture medium of sh-PRSS3 and sh-PRSS3+ov-NC groups,the viability of CD8+T cells was increased,the levels of IFN-γ,TNF-α,granzyme B and perforin were elevated,while the apoptosis of CD8+T cells and the level of sPD-L1 were decreased,as compared with sh-NC group.In the sh-PRSS3+ov-FGFR2 group,all these differences mentoned above were mitigated,which means the effects of sh-PRSS3 were antagonized by overexpression of FGFR2.Conclusion PRSS3 induces immune escape of NSCLC cells by up-regulating FGFR2 expression,and its mechanism may be related to the up-regulation of PD-L1 expression induced by activating JAK2/STAT3 signaling pathway.
维普
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