从真核生物到原核生物，所有细胞都会分泌细胞外囊泡 (EV)，作为其正常体内平衡、细胞间通讯和物质处理的一部分。越来越多的证据表明，小型 EV 携带功能性小 RNA，有可能充当细胞外信使和液体活检标记物。然而，由于严重的限制，包括用于测序的方案、短读长 (20-50 nt) 的次优比对以及未表征的基因组注释（通常表示为基因组的“暗物质”。在这项研究中，我们研究了与 EV 相关的未注释的小 RNA，这些 RNA 源自内源基因，是基因组“暗物质”的一部分，可能在调节基因表达和翻译机制中发挥关键的新兴作用。为了解决这个问题，我们创建了一个来自人类前列腺癌的独特的小型 RNAseq 数据集

From eukaryotes to prokaryotes, all cells secrete extracellular vesicles (EVs) as part of their regular homeostasis, intercellular communication, and cargo disposal. Accumulating evidence suggests that small EVs carry functional small RNAs, potentially serving as extracellular messengers and liquid-biopsy markers. Yet, the complete transcriptomic landscape of EV-associated small RNAs during disease progression is poorly delineated due to critical limitations including the protocols used for sequencing, suboptimal alignment of short reads (20-50 nt), and uncharacterized genome annotations-often denoted as the 'dark matter' of the genome. In this study, we investigate the EV-associated small unannotated RNAs that arise from endogenous genes and are part of the genomic 'dark matter', which may play a key emerging role in regulating gene expression and translational mechanisms. To address this, we created a distinct small RNAseq dataset from human prostate cancer & benign tissues, and EVs derived from blood (pre- & post-prostatectomy), urine, and human prostate carcinoma epithelial cell line. We then developed an unsupervised data-based bioinformatic pipeline that recognizes biologically relevant transcriptional signals irrespective of their genomic annotation. Using this approach, we discovered distinct EV-RNA expression patterns emerging from the un-annotated genomic regions (UGRs) of the transcriptomes associated with tissue-specific phenotypes. We have named these novel EV-associated small RNAs as 'EV-UGRs' or "EV-dark matter". Here, we demonstrate that EV-UGR gene expressions are downregulated by ∼100 fold (FDR < 0.05) in the circulating serum EVs from aggressive prostate cancer subjects. Remarkably, these EV-UGRs expression signatures were regained (upregulated) after radical prostatectomy in the same follow-up patients. Finally, we developed a stem-loop RT-qPCR assay that validated prostate cancer-specific EV-UGRs for selective fluid-based diagnostics. Overall, using an unsupervised data driven approach, we investigate the 'dark matter' of EV-transcriptome and demonstrate that EV-UGRs carry tissue-specific Information that significantly alters pre- and post-prostatectomy in the prostate cancer patients. Although further validation in randomized clinical trials is required, this new class of EV-RNAs hold promise in liquid-biopsy by avoiding highly invasive biopsy procedures in prostate cancer.© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.