Comprehensive Analysis of Serum Small Extracellular Vesicles-Derived Coding and Non-Coding RNAs from Retinoblastoma Patients for Identifying Regulatory Interactions

SIMPLE SUMMARY: The diagnosis of retinoblastoma (RB) is usually made by clinical examination and imaging modalities. Routine tissue biopsy is not done due to the risk of extraocular spread. Blood-based RNA cargoes could be promising surrogate markers for RB diagnosis and prognostication. Our data indicated that the size, morphology, and zeta potential (ZP) of RB and non-RB serum extracellular vesicles (EVs) met standard exosome properties with similar concentrations. MALTA1, AFAP1-AS1, miR-145, and miR-101 were identified as hub non-coding RNAs that promote RB progression by targeting cyclins, cyclin-dependent kinases, c-MYC, EZH2, ZEB1, TP53, and BCL2. Along with these, the aberrantly expressed miRNAs, lncRNAs, and their target mRNAs of RB EVs were implicated in cell cycle, metabolism, and tumor-associated signaling pathways. The differential expression of EV RNAs in RB compared to controls may aid in the identification of possible serum prognostic biomarkers for RB. ABSTRACT: The present study employed nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, RNA sequencing, and quantitative real-time PCR validation to characterize serum-derived small extracellular vesicles (sEVs) from RB patients and age-matched controls. Bioinformatics methods were used to analyze functions, and regulatory interactions between coding and non-coding (nc) sEVs RNAs. The results revealed that the isolated sEVs are round-shaped with a size < 150 nm, 5.3 × 10(11) ± 8.1 particles/mL, and zeta potential of 11.1 to −15.8 mV, and expressed exosome markers CD9, CD81, and TSG101. A total of 6514 differentially expressed (DE) mRNAs, 123 DE miRNAs, and 3634 DE lncRNAs were detected. Both miRNA-mRNA and lncRNA-miRNA-mRNA network analysis revealed that the cell cycle-specific genes including CDKNI1A, CCND1, c-MYC, and HIF1A are regulated by hub ncRNAs MALAT1, AFAP1-AS1, miR145, 101, and 16-5p. Protein-protein interaction network analysis showed that eye-related DE mRNAs are involved in rod cell differentiation, cone cell development, and retinol metabolism. In conclusion, our study provides a comprehensive overview of the RB sEV RNAs and regulatory interactions between them.