Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany.
Clinic of Dermatology, Universitäts-Spital Zürich, Zürich, Switzerland.
Clin Transl Med. 2022 Jul;12(7):e934. doi: 10.1002/ctm2.934.
Immune checkpoint inhibitors directed against programmed cell death 1 (PDCD1/PD1) receptor and programmed cell death-ligand 1 (CD274/PD-L1) have been recently successfully implemented for the treatment of many cancers, but the response rate of tumour patients is still limited due to intrinsic and acquired resistances. However, the underlying molecular mechanisms of this limited response have still to be defined in detail. The aim of this study is to uncover processes inhibiting PDCD1/CD274 expression thereby enhancing anti-tumour immune responses. The identification and characterization of microRNAs (miRNAs) targeting the 3'-untranslated region (3'-UTR) as well as the coding sequence (CDS) of CD274 will provide the basis for a new drug development.
Human melanoma cell lines and tissue samples were subjected to mRNA and/or protein expression analysis using qPCR, Western blot, flow cytometry, and/or immunohistochemistry. The data were correlated to clinical parameters. MiRNA trapping by RNA in vitro affinity purification (miTRAP) technology in combination with small RNA sequencing and different bioinformatics tools were employed to identify CD274-regulating miRNAs.
Screening based on miTRAP in combination with RNAseq identified a large number of novel CD274-regulating candidate miRNAs, from which eight selected miRNAs were functionally validated. Five out of eight miRNAs were able to significantly reduce CD274 surface expression indicating that these miRNAs directly bind to the 3'-UTR or CDS of the CD274 gene. The miRNA-mediated inhibition of CD274 expression was accompanied by an increased T cell recognition. Furthermore, an inverse expression of three CD274-regulating miRNAs and CD274 was demonstrated in melanoma lesions. A CD274 miRNA score was generated, which was associated with disease progression and reduced survival of melanoma patients.
These data revealed a novel mechanism that miRNAs targeting the CDS of immune checkpoint genes are functional, have prognostic relevance, and also the potential for the development of novel miRNA-based therapies.
针对程序性细胞死亡 1（PDCD1/PD1）受体和程序性细胞死亡配体 1（CD274/PD-L1）的免疫检查点抑制剂最近已成功用于治疗许多癌症，但由于内在和获得性耐药，肿瘤患者的反应率仍然有限。然而，这种有限反应的潜在分子机制仍有待详细定义。本研究旨在揭示抑制 PDCD1/CD274 表达从而增强抗肿瘤免疫反应的过程。鉴定和表征靶向 CD274 的 3'非翻译区（3'-UTR）和编码序列（CDS）的 microRNAs（miRNAs）将为新药物的开发提供基础。
使用 qPCR、Western blot、流式细胞术和/或免疫组织化学对人黑色素瘤细胞系和组织样本进行 mRNA 和/或蛋白表达分析。将数据与临床参数相关联。采用体外 RNA 亲和力纯化（miTRAP）技术联合小 RNA 测序和不同的生物信息学工具来鉴定 CD274 调节 miRNA。
基于 miTRAP 与 RNAseq 的筛选鉴定了大量新型 CD274 调节候选 miRNA，其中 8 个选定 miRNA 进行了功能验证。在 8 个 miRNA 中有 5 个能够显著降低 CD274 表面表达，表明这些 miRNA 直接结合到 CD274 基因的 3'-UTR 或 CDS。CD274 表达的 miRNA 介导抑制伴随着 T 细胞识别的增加。此外，在黑色素瘤病变中证明了三种 CD274 调节 miRNA 和 CD274 的表达呈负相关。生成了一个 CD274 miRNA 评分，该评分与疾病进展和黑色素瘤患者的生存时间缩短相关。
这些数据揭示了一种新的机制，即靶向免疫检查点基因 CDS 的 miRNA 是功能性的，具有预后相关性，并且也具有开发新型 miRNA 治疗方法的潜力。
