FOXJ2转录激活胃癌细胞PD-L1表达并抑制T细胞抗肿瘤免疫的研究

FOXJ2 Inhibits T Cells Anti-tumor Immunity Through Transcriptional Activation of PD-L1 Expression in Gastric Cancer Cells

[目的]研究免疫检查点PD-L1在胃癌细胞中的转录调控机制;探讨转录因子FOXJ2对PD-L1的转录调控,两者表达的相关性及其与胃癌患者预后的关系;揭示FOXJ2在调控T细胞抗肿瘤免疫中发挥的生物学功能。[方法]利用生物信息学方法筛选能够调控PD-L1的转录因子;免疫组化及免疫荧光检测FOXJ2和PD-L1在胃癌临床标本中的表达以及FOXJ2与CD8;T细胞浸润的关系;qRT-PCR与Western blot检测FOXJ2和PD-L1在胃癌细胞中的表达;ChIP实验验证FOXJ2与PD-L1基因启动子的直接相互作用;胃癌细胞与T细胞共培养实验检测T细胞对肿瘤细胞的杀伤能力;流式细胞术检测肿瘤细胞中PD-L1表达情况及T细胞中TNF-α和IFN-γ的表达情况。[结果]JASPAR和PROMO数据库筛选出24个可能调控PD-L1的候选转录因子;癌症公共数据库显示FOXJ2与PD-L1的表达呈正相关(P<0.05),高表达FOXJ2的胃癌患者生存期明显缩短(P<0.01)。FOXJ2在胃癌组织中的表达较癌旁组织显著上调,且与PD-L1的表达呈正相关关系(R=0.629,P<0.01)。qRT-PCR与Western blot证实胃癌细胞中敲低FOXJ2表达后PD-L1表达下降。ChIP实验证实FOXJ2与PD-L1基因启动子区域的直接相互作用。下调胃癌细胞中FOXJ2表达后,T细胞对肿瘤细胞的杀伤能力增强;流式细胞术显示肿瘤细胞中的PD-L1下降,而T细胞中TNF-α和IFN-γ的表达升高。[结论]FOXJ2与PD-L1表达和胃癌患者的生存密切相关,阻断FOXJ2对PD-L1的转录调控有望成为胃癌免疫治疗的新靶点。

[Purpose]To investigate the regulatory effect of FOXJ2 on PD-L1 expression in gastric cancer(GC).[Methods]Bioinformatics methods were performed to screen transcription factors for regulating PD-L1.The expression of FOXJ2 and PD-L1 in GC specimens was detected by qRT-PCR and Western blot.The relationship between FOXJ2 and CD8;T cell was explored by immunohistochemistry and immunofluorescence.The ChIP experiments were conducted to verify the interaction between FOXJ2 and PD-L1 promoter.Co-culturing GC cell and T cell were used to detect the killing ability of T cells on tumor cells.Flow cytometry was used to detect the expression of PD-L1 in tumor cells and the expression of TNF-αand IFN-γin T cells.[Results]JASPAR and PROMO databases predicted 24 candidate transcription factors that may regulate PD-L1.The cancer public databases showed that the expression of FOXJ2 and PD-L1 were positively correlated(P<0.05)in GC.The high expression of FOXJ2 correlated with poor GC patient prognosis(P<0.01).The tissue microarrays revealed that the expression of FOXJ2 in GC tissues was higher than that in adjacent tissues,and it was positively correlated with the expression of PD-L1(R=0.629,P<0.01).The expression of PD-L1 decreased if FOXJ2 was downregulated by siRNA in GC cells.ChIP experiments confirmed the direct interaction between FOXJ2 and PD-L1 gene promoter region.When FOXJ2 was downregulated,the killing ability of T cells on tumor cells was enhanced;the expression of PD-L1 decreased in tumor cells,whereas TNF-αand IFN-γincreased in T cells.[Conclusion]FOXJ2 is closely related to the survival and prognosis of patients with gastric cancer,and blocking the transcriptional regulation of FOXJ2 on PD-L1 is expected to be a new immunotherapy target in GC.