Comprehensive profiling of extracellular RNA in HPV-induced cancers using an improved pipeline for small RNA-seq analysis

Extracellular RNAs (exRNAs) have attracted great attention due to their essential role in cell-to-cell communication as well as their potential as non-invasive disease biomarkers. However, at present, there is no consensus on the best method to profile exRNA expression, which leads to significant variability across studies. To address this issue, we established an experimental pipeline for comprehensive profiling of small exRNAs isolated from cell culture. By evaluating six RNA extraction protocols, we developed an improved method for robust recovery of vesicle-bound exRNAs. With this method, we performed small RNA sequencing of exosomes (EXOs), microvesicles (MVs) and source cells from 14 cancer cell lines. Compared to cells, EXOs and MVs were similarly enriched in tRNAs and rRNAs, but depleted in snoRNAs. By miRNA profiling analysis, we identified a subset of miRNAs, most noticeably miR-122-5p, that were significantly over-represented in EXOs and MVs across all 14 cell lines. In addition, we also identified a subset of EXO miRNAs associated with cancer type or human papillomavirus (HPV) status, suggesting their potential roles in HPV-induced cancers. In summary, our work has laid a solid foundation for further standardization on exRNA analysis across various cellular systems.