Research Group GMP & T Cell Therapy, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany.
Clin Transl Med. 2023 Feb;13(2):e1186. doi: 10.1002/ctm2.1186.
The regulatory functions of microRNAs (miRNAs) in anti-tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell-target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8 T cell-mediated cytotoxicity.
Luciferase expressing B16F10 melanoma (B16F10 Luci ) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma-associated antigen tyrosinase-related protein 2. miRNAs with the most pronounced effects on T cell-mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)-mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA-seq) data from miRNA-overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)-derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients' survival.
The miRNA screen resulted in the selection of seven miRNAs enhancing CTL-mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa-miR-320a-3p, mmu-miR-7037-5p and mmu-miR-666-3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA-mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA-seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA-mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined.
For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell-mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.
微小 RNA（miRNAs）在抗肿瘤免疫中的调控作用主要在免疫效应细胞中描述。由于人们对 T 细胞-靶细胞相互作用过程中靶细胞易感性的 miRNA 影响知之甚少，因此本研究侧重于鉴定在肿瘤细胞中表达的 miRNA，以控制其对 CD8 T 细胞介导的细胞毒性的易感性。
用涵盖综合小鼠 miRNA 文库的单个 miRNA 转染表达荧光素酶的 B16F10 黑色素瘤（B16F10 Luci）细胞，筛选对黑色素瘤相关抗原酪氨酸酶相关蛋白 2 特异性的已建立的细胞毒性 T 淋巴细胞（CTL）系（5a，克隆 Nβ）裂解的易感性。对最显著影响 T 细胞介导的裂解的 miRNA 进行验证，并在 B16F10 细胞中稳定表达。通过筛选确定 miRNA 集合的共同靶点的计算机分析，并用调节免疫监视的小干扰 RNA（siRNA）介导的沉默实验进一步证实。应用 IPA 软件和 miRNA 过表达细胞系的 RNA 测序（RNA-seq）数据来研究潜在的机制。利用癌症基因组图谱（TCGA）衍生的 miRNA 测序数据来评估 miRNA 表达与黑色素瘤患者生存的相关性。
miRNA 筛选导致七种 miRNA 体外增强 CTL 介导的黑色素瘤细胞杀伤。在选定的 miRNA 稳定过表达后，hsa-miR-320a-3p、mmu-miR-7037-5p 和 mmu-miR-666-3p 被确定为增强 CTL 溶解的最有效 miRNA。计算机分析和随后的 siRNA 介导的沉默实验鉴定 Psmc3 和 Ndufa1 为可能参与观察到的功能效应的共同 miRNA 靶标。IPA 对 RNA-seq 数据分析显示，潜在参与 miRNA 介导功能效应的途径、网络、生物学功能和关键分子。最后，基于 TCGA 数据分析，确定在鉴定的七种 miRNA 中保守 miRNA 与黑色素瘤患者总生存的正相关性。
本研究首次发现了影响黑色素瘤细胞对 T 细胞介导杀伤易感性的 miRNA 种类。这些 miRNA 可能是针对黑色素瘤和其他肿瘤实体的新型治疗方法的有吸引力的候选物。
