内质网应激头颈部鳞状细胞癌细胞通过外泌体miR-26a-5p调控PTEN/AKT通路促进巨噬细胞PD-L1表达

Endoplasmic reticulum stressed HNSCC cell-derived exosomal miR-26a-5p promotes PD-L1 expression in macrophage through PTEN/AKT signaling pathway

摘要

目的 探讨内质网应激（endoplasmic reticulum stress，ERS）的头颈部鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）细胞分泌的外泌体miRNA表达变化对巨噬细胞的影响机制。方法 本研究已通过单位伦理委员会审查批准。通过蛋白质免疫印迹（Western blot，WB）和实时荧光定量PCR实验（RT-qPCR）检测HNSCC肿瘤组织及癌旁组织ERS相关蛋白，包括蛋白激酶R样内质网激酶（protein kinase R-like endoplasmic reticulum kinase，PERK）和葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）的表达水平；以500 U/mL干扰素-γ（interferon-γ，IFN-γ）处理人喉鳞癌细胞系HN4细胞48 h，诱发HN4细胞产生内质网应激反应，收集HN4细胞分泌的外泌体，通过生物信息学分析鉴定外泌体的miRNA种类，预测miRNA的靶基因，将巨噬细胞转染miRNA、与收集的外泌体共培养、并敲低巨噬细胞PTEN表达，以WB和RT-qPCR检测外泌体miRNA调控的下游信号通路蛋白酪氨酸磷酸酶（phosphate and tension homology deleted on chromsome ten，PTEN）/蛋白激酶B（protein kinase B，AKT）及程序性死亡受体-1配体（programmed death receptor ligand 1，PD-L1）表达变化。结果 HNSCC肿瘤组织相比癌旁组织ERS相关蛋白表达水平升高（P<0.05）；RNA测序及实验验证表明ERS的HN4细胞分泌的外泌体miR-26a-5p表达上调（P<0.05）；PTEN是miR-26a-5p的靶基因，miR-26a-5p升高巨噬细胞PD-L1表达水平，并下调PTEN表达（P<0.05）；巨噬细胞与ERS外泌体共培养，miR-26a-5p和PD-L1表达上升，PTEN表达下降，p-AKT表达升高（P<0.05）；敲低巨噬细胞PTEN表达，PD-L1表达上升（P<0.01）。结论 ERS的HNSCC细胞通过外泌体miR-26a-5p调控PTEN/AKT通路及PD-L1的表达。

Abstract

Objective To investigate the impact of exosomal miRNAs derived from endoplasmic reticulum-stressed (ERS) head and neck squamous cell carcinoma (HNSCC) cells on macrophages. Methods This study was reviewed and approved by the Ethics Committee. The expression levels of ERS-associated proteins, including protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78), in HNSCC tissues and para-tumor tissues were detected by Western blot (WB) and quantitative real-time PCR (RT-qPCR). HN4 human laryngeal squamous cell carcinoma cells were treated with 500 U/mL interferon-γ (IFN-γ) for 48 h to induce ER stress, and exosomes secreted by ER-stressed HN4 cells were collected and identified. The types of miRNAs in exosomes were identified through bioinformatics analysis, and the target genes of miRNAs were predicted. Macrophages were transfected with miRNA, co cultured with collected exosomes, and the expression of PTEN in macrophages was knocked down. The downstream signaling pathway regulated by exosomal miRNAs was studied by WB and RT-qPCR. Results Compared with that in para-tumor tissues, the expression level of ER stress-associated proteins in HNSCC tissues was increased (P<0.05). RNA-seq analysis revealed that miR-26a-5p was highly upregulated in ER-stressed HN4 cell-derived exosomes (P<0.05). PTEN is the target gene for miR-26a-5p. miR-26a-5p increased the expression level of PD-L1 in macrophages and downregulated the expression of PTEN (P<0.05). Macrophages co cultured with ERS extracellular vesicles showed an increase in miR-26a-5p and PD-L1 expression, a decrease in PTEN expression, and an increase in p-AKT expression (P<0.05). Knock down the expression of PTEN in macrophages and increase the expression of PD-L1 (P<0.01). Conclusion ERS HNSCC cell-derived exosomal miR-26a-5p promotes the expression of PD-L1 in macrophages through the PTEN/AKT signaling pathway.

关键词

头颈部鳞状细胞癌 / 内质网应激 / miR-26a-5p / 外泌体 / 巨噬细胞 / 程序性死亡受体-1配体 / 蛋白酪氨酸磷酸酶 / 蛋白激酶B

Key words

head and neck squamous cell carcinoma / endoplasmic reticulum stress / exosome / miR-26a-5p / macrophage / programmed death receptor ligand 1 / phosphate and tension homology deleted on chromsome ten / protein kinase B

Author summay

焦鹏飞，住院医师，硕士，Email：[e-mail]