环状 RNA 0000512 通过海绵吸附 miR-622/CMTM6 轴抑制 PD-L1 泛素化，促进三阴性乳腺癌和免疫逃逸。
circ-0000512 inhibits PD-L1 ubiquitination through sponging miR-622/CMTM6 axis to promote triple-negative breast cancer and immune escape.
机构信息
Department of Breast Surgery and Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People's Republic of China
Department of Breast Surgery, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People's Republic of China.
出版信息
J Immunother Cancer. 2023 Jun;11(6). doi: 10.1136/jitc-2022-005461.
BACKGROUND
This study reported the function and mechanism of circ-0000512 in the progression of triple-negative breast cancer (TNBC).
METHODS
circ-0000512 expression in TNBC tissues and paired adjacent normal tissues and cells was examined by qRT-PCR. Moreover, circ-0000512 expression in TNBC cells was modulated by transfection. Thereafter, colony formation assay, Transwell assay and flow cytometry were conducted to observe cell proliferation, migration and apoptosis. TNBC cells were treated with cycloheximide and the protease inhibitor MG132. Later, ubiquitination assay was performed to detect programmed cell death ligand 1 (PD-L1) ubiquitination in TNBC cells. The T cell killing ability was assessed by the T cell-mediated tumor cell killing assay. IFNγ and IL-2 levels were detected by ELISA. The percentage of activated T cells was detected with a flow cytometer. In addition, dual luciferase reporter gene assay and RNA immunoprecipitation assay were carried out to evaluate the binding between two genes. In vivo study was conducted on mice. CD8+ T cells in xenograft tumors were detected by immunohistochemistry.
RESULTS
circ-0000512 was upregulated in patients with TNBC. circ-0000512 knockdown attenuated the proliferation and migration of TNBC cells and enhanced their apoptosis. circ-0000512 overexpression had opposite effects. circ-0000512 knockdown enhanced the PD-L1 protein ubiquitination in TNBC cells by inhibiting CMTM6. Meanwhile, circ-0000512 promoted CMTM6 expression by sponging miR-622. circ-0000512 knockdown increased the ratio of CD8+T cells and the lethality of T cells against TNBC cells. Besides, circ-0000512 knockdown inhibited the growth of TNBC cells in immunodeficient nude mice and normal immune mice and increased the ratio of CD8+T cells in xenograft tumors of normal immune mice.
CONCLUSIONS
circ-0000512 inhibited PD-L1 ubiquitination by sponging the miR-622/CMTM6 axis, thus promoting TNBC progression and immune escape.
背景
本研究报道了 circ-0000512 在三阴性乳腺癌（TNBC）进展中的作用和机制。
方法
通过 qRT-PCR 检测 TNBC 组织和配对相邻正常组织及细胞中的 circ-0000512 表达。此外，通过转染调节 TNBC 细胞中的 circ-0000512 表达。然后，进行集落形成实验、Transwell 实验和流式细胞术观察细胞增殖、迁移和凋亡。用环己酰亚胺和蛋白酶抑制剂 MG132 处理 TNBC 细胞。随后，进行泛素化实验检测 TNBC 细胞中程序性细胞死亡配体 1（PD-L1）的泛素化。通过 T 细胞介导的肿瘤细胞杀伤实验评估 T 细胞的杀伤能力。通过 ELISA 检测 IFNγ 和 IL-2 水平。用流式细胞仪检测活化 T 细胞的百分比。此外，进行双荧光素酶报告基因实验和 RNA 免疫沉淀实验评估两个基因之间的结合。在小鼠中进行体内研究。用免疫组化检测异种移植肿瘤中的 CD8+T 细胞。
结果
circ-0000512 在 TNBC 患者中上调。circ-0000512 敲低可减弱 TNBC 细胞的增殖和迁移，增强其凋亡。circ-0000512 过表达则产生相反的效果。circ-0000512 敲低通过抑制 CMTM6 抑制 TNBC 细胞中 PD-L1 蛋白的泛素化。同时，circ-0000512 通过海绵吸附 miR-622 促进 CMTM6 的表达。circ-0000512 敲低增加了 CD8+T 细胞的比例和 T 细胞对 TNBC 细胞的杀伤能力。此外，circ-0000512 敲低抑制免疫缺陷型裸鼠和正常免疫小鼠中 TNBC 细胞的生长，增加正常免疫小鼠异种移植肿瘤中 CD8+T 细胞的比例。
结论
circ-0000512 通过海绵吸附 miR-622/CMTM6 轴抑制 PD-L1 泛素化，从而促进 TNBC 的进展和免疫逃避。
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