Chen Tao, Dong Yaping, Wu Xiaoxue
Clinical Laboratory, The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250001, China.
Oncology Department, The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250001, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022 Apr;38(4):347-356.
Objective To explore the effects of miR-335-5p derived from plasma exosomes on immune escape of triple-negative breast cancer (TNBC) via regulating ubiquitin-specific protease 22 (USP22). Methods The plasma of TNBC patients and healthy people was collected, then plasma exosomes were separated, and real time quantitative PCR was used to determine the relative expression of miR-335-5p in exosomes. The interaction between miR-335-5p and USP22 was verified by dual luciferase reporter assay. The expression of miR-335-5p and USP22 in exosomes and MDA-MB-436 cells was regulated. Exosomes or MDA-MB-436 cells were co-cultured with CD8 T lymphocytes and subsequently divided into different groups.The apoptosis of cells in each group was detected by flow cytometry, and the levels of interferon γ (IFN-γ) and tumor necrosis factor α (TNF- α) in each group were detected by ELISA. The effects of USP22 on the stability of programmed death 1 ligand 1(PD-L1) was tested by Western blot analysis. The effects of miR-335-5p and PD-L1 on tumor growth was detected by tumor formation test in nude mice. Results The expression of miR-335-5p in TNBC exosomes was down-regulated. USP22 was confirmed as a target gene of miR-335-5p. In addition, USP22 could inhibit the ubiquitination of PD-L1 protein. Overexpression of miR-335-5p inhibited the immune escape of TNBC. Inhibition of miR-335-5p promoted the immune escape of TNBC, which could be partially saved by USP22 down-regulation. Knockdown of miR-335-5p promoted tumor growth in vivo, while tumor growth was inhibited by the addition of PD-L1 antibody. Conclusion Exosomal miR-335-5p promotes ubiquitination of PD-L1 by USP22 through down-regulating USP22, and inhibits TNBC immune escape mediated by PD-L1.
目的 探讨血浆外泌体来源的miR-335-5p通过调控泛素特异性蛋白酶22（USP22）对三阴性乳腺癌（TNBC）免疫逃逸的影响。方法 收集TNBC患者及健康人的血浆，分离血浆外泌体，采用实时定量PCR检测外泌体中miR-335-5p的相对表达量。通过双荧光素酶报告基因检测验证miR-335-5p与USP22的相互作用。调控外泌体及MDA-MB-436细胞中miR-335-5p和USP22的表达。将外泌体或MDA-MB-436细胞与CD8 T淋巴细胞共培养，随后分为不同组。采用流式细胞术检测各组细胞凋亡情况，采用酶联免疫吸附测定（ELISA）检测各组干扰素γ（IFN-γ）和肿瘤坏死因子α（TNF-α）水平。通过蛋白质免疫印迹分析检测USP22对程序性死亡受体1配体1（PD-L1）稳定性的影响。通过裸鼠成瘤实验检测miR-335-5p和PD-L1对肿瘤生长的影响。结果 TNBC外泌体中miR-335-5p表达下调。USP22被确认为miR-335-5p的靶基因。此外，USP22可抑制PD-L1蛋白的泛素化。miR-335-5p过表达抑制TNBC的免疫逃逸。抑制miR-335-5p促进TNBC的免疫逃逸，下调USP22可部分挽救该作用。敲低miR-335-5p促进体内肿瘤生长，而添加PD-L1抗体可抑制肿瘤生长。结论 外泌体miR-335-5p通过下调USP22促进PD-L1的泛素化，抑制由PD-L1介导的TNBC免疫逃逸。
