Project description:To check the profile of exosomal and cellular miRNA in ovarian cancer cell lines, total RNA were extracted from exosomes and cells. Thirteen ovarian cancer cell lines (A2780, ES-2, CAOV3, SKOV3, OV-90, OAW42, MCAS, COV362, RMG-1, RMUG-S, KURAMOCHI, NIH-OVCAR3 and A2780cis) were investigated, and HOSE1, HOSE2 and HOSE3 (human ovarian surface epithelim cell lines) were used as control.
Project description:The exosomal particles in the blood of cancer patients possess many times more tumor markers than free circulating. Among these elements, miRNAs have great biomedical relevance due to their stability and feasible detection. However, there is not available in the market any reliable endogenous control for the exosomal compartment, nor specific for the miRNA content, preventing the obtainment of standardization measures in cancer liquid-biopsy. In this study, we firstly identified three miRNAs out of a panel of nine potential normalizers that arised from an integrative analysis comparing the global miRNA exosomal profile by miRNA-seq of six lung and ovarian human cancer cell lines under different chemotherapy conditions. Their value as normalizers were also tested in 14 additional human cancer cell lines from different tumor types and after radiotherapy and an alternative chemotherapy treatment. Its translational validation comprised 124 prospective samples, 70 plasma from NSCLC patients, 12 from glioblastoma patients, 10 healthy donors and 16 paired samples from plasma and ascites fluid from ovarian cancer patients. The variability and normalizing properties were tested in comparison with the tissue-origin gold standard miR-16. Our results indicate that miR-151a is constantly represented in the exosomal content with minimal variability compared with miR16 independently of many conditions tested, providing for the first time a universal normalizer for the exosomal compartment that will impact the use of liquid biopsy.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes