照射诱导自噬对三阴性乳腺癌中PD-L1介导免疫抑制的影响

Effect of Radiation Induced Autophagy on PD-L1 Mediated Immunosuppression in Triple Negative Breast Cancer

关键词:  照射  外泌体  自噬  PD-L1  免疫调控

Key words:  irradiation  exosomes  autophagy  PD-L1  immune regulation

摘要:

三阴性乳腺癌(Triple Negative Breast Cancer，TNBC)中外泌体(Exosomes)携带的细胞程序性死亡-配体1 (Programmed Cell Death Ligand 1，PD-L1)与肿瘤微环境(Tumor Microenvironment，TME)免疫抑制相关。为探讨照射能否调节PD-L1的表达从而改善TME的作用，本研究首先通过RT-qPCR和Western blot检测自噬抑制剂氯喹(Chloroquine,CQ)、自噬激活剂雷帕霉素(Rapamycin,Rapa)及蛋白酶体抑制剂MG132在联合或不联合照射下PD-L1、LC3B和P62蛋白的表达情况；其次，采用细胞免疫荧光检测照射前后PD-L1的表达及定位改变；再次，采用Western blot检测照射前后外泌体中PD-L1 (sEVs-PD-L1)的表达；最后，采用生物信息学对ATG7与乳腺癌预后、信号通路的相关性进行分析。结果显示：PD-L1在TNBC中的表达高于非三阴性乳腺癌(non-TNBC)；PD-L1可通过自噬-溶酶体降解，照射后诱导自噬可以促进PD-L1的胞浆转移及下调sEVs-PD-L1的表达，具体机制可能与信号转导和转录激活因子3 (Signal Transducer and Activator of Transcription 3,STAT3)的激活及外泌体转运相关。上述结果说明照射可以通过影响自噬及外泌体的发生，使PD-L1在微环境中表达下调，进而调控肿瘤介导的免疫抑制。

Abstract:

Programmed Cell Death Ligand 1 (PD-L1) carried by exosomes in Triple Negative Breast Cancer (TNBC) is associated with immunosuppression of Tumor Microenvironment (TME).In order to explore whether irradiation can regulate the expression of PD-L1 and improve the effect of TME,RT-qPCR and Western blot were used to detect the expression of PD-L1,LC3B,and P62 proteins in autophagy inhibitor Chloroquine (CQ),autophagy activator Rapamycin (Rapa),and proteasome inhibitor MG132 under combined or non combined irradiation.Secondly,the expression and localization of PD-L1 before and after irradiation were detected by immunofluorescence.Thirdly,the expression of PD-L1 (sEVs-PD-L1) in exosomes before and after irradiation was detected by Western blot.Finally,bioinformatics was used to analyze the correlation between ATG7 and breast cancer prognosis and signaling pathways.The results showed that the expression of PD-L1 in TNBC was higher than that in non-TNBC.PD-L1 can be degraded by autophagy and lysosome,while autophagy induction after irradiation can promote the cytoplasmic transfer of PD-L1 and down-regulate the expression of sEVs-PD-L1.The specific mechanism may be related to the activation of Signal Transducer and Activator of Transcription 3 (STAT3) and exosome transport.The above results show that irradiation can down-regulate the expression of PD-L1 in the microenvironment by affecting the occurrence of autophagy and exosomes,thereby regulating tumor-mediated immunosuppression.