RESUMEN
Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.
RESUMEN
As Vesículas Extracelulares (EVs) são organelas essenciais para processos fisiológicos e aparentemente contribuem para o desenvolvimento de diversas doenças através da transferência entre células de ácidos nucléicos e proteínas contidos em seu interior. Deste modo, EVs são promissoras como fonte de biomarcadores. Neste trabalho, avaliamos o conteúdo de EVs secretadas in vitro e in vivo, com o intuito de identificar moléculas relacionadas com a amplificação do oncogene ERBB2/HER2 e com estádios de progressão tumoral de câncer de mama HER2+. Utilizando um modelo de duas linhagens celulares - HB4a derivada de mama normal e C5.2, um clone de HB4a que superexpressa o oncogene ERBB2/HER2 - isolamos duas populações de EVs provenientes do meio de cultura celular condicionado: 20K, enriquecida para microvesículas e 100K, enriquecida para exossomos. Demonstramos que a superexpressão de um único oncogene (ERBB2/HER2) altera o perfil proteômico das EVs secretadas pela células C5.2. Proteínas capazes de induzir a transformação maligna foram encontradas superexpressas nas duas populações de EVs da linhagem C5.2, e interessantemente observamos uma maior expressão de HER2 nas EVs em relação a esta mesma linhagem celular. Uma análise por sequenciamento de nova geração do transcriptoma completo (RNAs pequenos e longos) das EVs e células secretoras revelou centenas de transcritos diferencialmente representados entre as duas linhagens, que podem estar direta ou indiretamente sendo afetados pela superexpressão de HER2, e possibilitou uma investigação das moléculas preferencialmente incorporadas nas EVs...
Extracellular vesicles (EVs) are essential organelles involved in physiological processes and apparently contribute to the development of several pathological conditions by the transfer between cells of nucleic acid and protein cargo. Therefore, EVs are promising sources of biomarkers. In the study presented here, we evaluated the content of EVs secreted in vitro and in vivo, aiming to identify molecules related to ERBB2/HER2 oncogene amplification and with progressing tumor stages of HER2+ breast cancer. Using a model of two cell lines HB4a derived from normal breast and C5.2, a clone of HB4a that overexpresses ERBB2/HER2 we isolated two populations of EVs derived from the conditioned cell culture medium: 20K, enriched for microvesicles and 100K, enriched for exosomes. We demonstrate that the overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of EVs secreted by C5.2 cells. Proteins capable of inducing malignant transformation were found overexpressed in both EVs populations secreted by C5.2 cells, and interestingly, we observed an increased expression of HER2 in the EVs compared to the secreting cells. Next generation sequencing analysis of the entire transcriptome (long and short RNAs) of EVs and secreting cells revealed hundreds of transcripts differentially expressed between the two cell lines, which may be directly or indirectly affected by HER2 overexpression, and enabled the investigation of molecules that are preferentially shuttled in EVs...