Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells – evidence of unique microRNA cargos

<b>Background</b>: Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos.<b>Methods</b>: To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform.<b>Results</b>: Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated transfer RNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with mi RNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi.<b>Conclusions</b>: This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.

<b>背景</b>：黑色素瘤细胞可向细胞外微环境释放多种类型的细胞外囊泡（extracellular vesicles, EVs），此类囊泡参与肿瘤微环境中的细胞通讯与信号转导过程。细胞外囊泡的亚型包括外泌体（exosomes）、微囊泡（microvesicles）与凋亡小体（apoptotic bodies），其携带蛋白质与遗传物质（RNA）载荷。<b>方法</b>：为明确黑色素瘤细胞来源的细胞外囊泡的RNA载荷的生物学贡献，我们采用小RNA测序技术鉴定不同细胞外囊泡亚型中的小RNA。通过经过验证的离心分离方案，我们从黑色素瘤细胞系MML-1中分离得到上述细胞外囊泡亚型，并利用Ion Torrent平台完成RNA测序。<b>结果</b>：我们在各细胞外囊泡亚型中检测到多种不同的非编码RNA，包括微小RNA（microRNA, miRNA）、线粒体相关转运RNA、小核仁RNA（small nucleolar RNA, snoRNA）、小核RNA（small nuclear RNA, snRNA）、Ro相关YRNA、穹隆RNA（vault RNA）以及YRNA。本研究在细胞及各细胞外囊泡亚型中共鉴定到1041种微小RNA。层级聚类分析显示，外泌体中富集了特定的微小RNA，包括hsa-miR-214-3p、hsa-miR-199a-3p与hsa-miR-155-5p，这些RNA均与黑色素瘤进展密切相关。将外泌体微小RNA与临床黑色素瘤样本中的微小RNA进行比对后发现，外泌体中的多种微小RNA也特异性表达于黑色素瘤组织中，而非良性痣组织。<b>结论</b>：本研究首次证实黑色素瘤细胞释放的细胞外囊泡亚型中存在不同种类的小RNA，且此类RNA与临床黑色素瘤组织具有显著的相似性，为解析细胞外囊泡相关的细胞外RNA在癌症中的作用提供了独到的见解。