Clinical relevance of exosome-derived microRNAs in Ovarian Cancer: Looking for new tumor biological fingerprints.

Specific tumor-derived extracellular vesicles, called exosomes, are considered as potential key players in cross-talk between immune system and tumor microenvironment in several solid tumors. Different studies highlighted the clinical relevance of exosomes in ovarian cancer (OC) for their role in early diagnosis, prognosis, chemoresistance, targeted therapy. The exosomes are nanosize vesicles carrying lipids, proteins, and nucleic acids. In particular, exosomes shuttle a wide spectrum of microRNAs (miRNAs) able to induce phenotypic reprogramming of target cells, contributing to tumor progression. In this review, we will discuss the promising role of miRNAs shuttled by exosomes, called exosomal miRNAs (exo-miRNAs), as potential biomarkers for early detection, tumour progression and metastasis, prognosis, and response to therapy in OC women, in order to search for new potential biological fingerprints able to better characterize the evolution of this malignancy and provide a clinically relevant non-invasive approach useful for adopting, in future, personalized therapeutic strategies.

In silico analysis of prognostic and diagnostic significance of target genes from prostate cancer cell lines derived exomicroRNAs.

BACKGROUND: Cancer-secreted exovesicles are important for cell-to-cell communication by altering cancer-related signalling pathways. Exovesicles-derived miRNAs (exomiRNAs)-target genes can be useful for diagnostic and prognostic purposes. METHODS: ExomiRNA from prostate cancer (PCa) cells (PC-3 and LNCaP) were quantified by qRT-PCR and compared to the healthy cell line RWPE-1 by using miRNome PCR 752 miRNAs Panel. MiRNet database was used to predict exomiRNA-target genes. ExomiRNA-target genes pathway functional enrichment was performed by using Reactome database and Enrichr platform. Protein-protein interaction analysis was carried out by using the STRING database. RNA target-gene sequencing data from The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) database was screened out in 465 PCa patients for candidate gene expression in prostate tumour (PT) tissue and non-pathologic prostate (N-PP) tissue. Signature gene candidates were statistically analysed for diagnosis and prognosis usefulness. RESULTS: A total of 36 exomiRNAs were found downregulated when comparing PCa cells vs a healthy cell line; and when comparing PC-3 vs LNCaP, 14 miRNAs were found downregulated and 52 upregulated. Reactome pathway database revealed altered pathways and genes related to miRNA biosynthesis, miRNA-mediated gene silencing (TNRC6B and AGO1), and cell proliferation (CDK6), among others. Results showed that TNRC6B gene expression was up-regulated in PT tissue compared to N-PP (n = 52 paired samples) and could be useful for diagnostic purposes. Likewise, gene expression levels of CDK6, TNRC6B, and AGO1 were down-regulated in high-risk PT (n = 293) compared to low-risk PCa tissue counterparts (n = 172). When gene expression levels of CDK6, TNRC6B, and AGO1 were tested as a prognostic panel, the results showed that these improve the prognostic power of classical biomarkers. CONCLUSION: ExomiRNAs-targets genes, TNRC6B, CDK6, and AGO1, showed a deregulated expression profile in PCa tissue and could be useful for PCa diagnosis and prognosis.

Exosomal MicroRNAs: Comprehensive Methods from Exosome Isolation to miRNA Extraction and Purity Analysis.

Exosomes are extracellular vesicles secreted by cells with a key role in a wide range of biological processes including cancer. These vesicles are involved in intercellular communication and deliver diverse cargo molecules, including miRNAs (exo-miRNAs), to recipient cells affecting their physiology. Exo-miRNAs have a role in promoting tumor, progression, metastatization, and remodeling of tumor microenvironment, therefore making them interesting biomarkers to study.Here we provide a detailed technical protocol for exosome isolation (which can be applied to cell culture as well as physiological fluids), validation of their vesicular identity, miRNA extraction, and quantitative and qualitative analysis to evaluate the sample purity and concentration.

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

A Serum Biomarker Panel of exomiR-451a, exomiR-25-3p and Soluble TWEAK for Early Diagnosis of Rheumatoid Arthritis.

Background: The etiology of rheumatoid arthritis (RA) remains poorly understood. Early and accurate diagnosis still difficult to achieve. Inflammatory related molecules released into the circulation such cytokines and exosome-derived microRNAs (exomiRNAs) could be good candidates for early diagnosis of autoimmune diseases. We sought to discover a serum biomarker panel for the early detection of RA based on exomiRNAs and inflammatory markers. Methods: A 179 miRNAs-microarray panel was analyzed in a pilot study (4 early RA and 4 controls). Validation of deregulated exomiRNAs was performed in a larger cohort (24 patients with early RA and 24 controls). miRNet software was used to predict exomiRNA gene-targets interactions. Potentially altered pathways were analyzed by Reactome pathway database search. STRING database was used to predict protein-protein interaction networks. Enzyme-linked immunosorbent assay was used to measure serum levels of sTWEAK and sCD163. Signature biomarker candidates were statistical analyzed. Results: We detected 11 differentially expressed exomiRNAs in early RA pilot study. Validation analysis revealed that 6/11 exomiRNAs showed strong agreement with the pilot microarray data (exomiR-144-3p, -25-3p, -15a-5p, -451a, -107 and -185-5p). sTWEAK and sCD163 biomarkers were significantly elevated in the serum of patients with early RA. Receiver operating characteristic (ROC) analysis showed that the best panel to diagnose early RA contained exomiR-451a, exomiR-25-3p and sTWEAK, and could correctly classify 95.6% of patients, with an area under the ROC curve of 0.983 and with 100% specificity and 85.7% sensitivity. The YWHAB gene was identified as a common target of the putative miRNA-regulated pathways. Conclusion: A novel serum biomarker panel composed of exomiR-451a, exomiR-25-3p and serum levels of sTWEAK may have use in the early clinical diagnosis of RA. A new predicted exomiRNA-target gene YHWAB has been identified and may have a relevant role in the development of RA.