Introduction:The interface between T cells and the tumor microenvironment, termed the ‘immunological synapse’, consists of multiple checkpoint protein pairs co-expressed on both sides of the synapse. mir-16, a microRNA from a widely known tumor-suppressor family of miRNAs, was previously shown by us to be downregulated in melanoma. As other miRNAs from this family have been shown to directly target checkpoint proteins, here we investigated whether miR-16 influences the expression patterns of checkpoint proteins in melanoma.Methods:Single-cell gene expression data from the melanoma microenvironment were retrieved from a public database. Melanoma cell lines were established from metastatic lesions and transiently transfected with an hsa-miR-16-5p-mimic RNA or a mir-16-expressing plasmid. The mRNA expression profiles were analyzed using an Affymetrix microarray. Direct targets of miR-16 were identified by luciferase reporter assays. Protein levels were assessed by Western blotting.Results:Bioinformatic analysis revealed that the expression levels of eight checkpoint mRNAs, known to be present on the melanoma side of the immunological synapse, were highly correlated. Four of these mRNAs contained putative binding sites for the miR-15/16 family. miR-16 expression was significantly reduced in melanoma cells, compared to normal melanocytes. Luciferase reporter assays demonstrated that miR-16 directly targets the 3′ untranslated regions (3′UTRs) of CD40, CD80. The mRNAs downregulated following miR-16 overexpression were highly enriched for genes involved in autophagy, vesicle-mediated transport, and the regulation of protein membrane localization. Among these, VTI1B and SMPD1 were confirmed to be direct targets of miR-16. Transient overexpression of miR-16 resulted in a significant reduction in SMPD1 and VTI1B levels in melanoma cell lines.Conclusions:Our findings suggest that miR-16 potentially modulates melanoma tumorigenesis, metastasis and immunogenicity by altering the composition of checkpoint proteins at the immunological synapse and by regulating cellular pathways associated with intracellular trafficking and transmembrane protein presentation.
引言：T细胞与肿瘤微环境之间的界面被称为"免疫突触"，由突触两侧共表达的多种检查点蛋白对构成。mir-16作为广为人知的抑癌miRNA家族成员，我们先前研究发现其在黑色素瘤中表达下调。鉴于该家族其他miRNA已被证实可直接靶向检查点蛋白，本研究旨在探讨miR-16是否影响黑色素瘤中检查点蛋白的表达模式。 方法：从公共数据库获取黑色素瘤微环境的单细胞基因表达数据。通过转移性病灶建立黑色素瘤细胞系，并瞬时转染hsa-miR-16-5p模拟RNA或mir-16表达质粒。采用Affymetrix基因芯片分析mRNA表达谱，通过荧光素酶报告基因实验鉴定miR-16的直接靶标，蛋白质水平通过Western blotting进行检测。 结果：生物信息学分析显示，已知存在于免疫突触黑色素瘤侧的8种检查点mRNA表达水平呈高度相关性，其中4种含有miR-15/16家族的潜在结合位点。与正常黑素细胞相比，黑色素瘤细胞中miR-16表达显著降低。荧光素酶报告实验证实miR-16可直接靶向CD40、CD80的3′非翻译区。miR-16过表达后下调的mRNA在自噬、囊泡介导运输及蛋白膜定位调控相关基因中高度富集，其中VTI1B和SMPD1被确认为miR-16的直接靶标。瞬时过表达miR-16可显著降低黑色素瘤细胞系中SMPD1和VTI1B水平。 结论：本研究提示miR-16可能通过改变免疫突触检查点蛋白组成，并调控细胞内运输和跨膜蛋白呈递相关通路，从而影响黑色素瘤的肿瘤发生、转移及免疫原性。
肿瘤(癌症)患者之家