Comparison of Methods for MicroRNA Isolation from Extracellular Vesicles Obtained from Ascitic Fluids.

Secreted extracellular vesicles (EVs) contain active biomolecules, including miRNAs, composition of which reflects epigenetic changes occurring in cells during pathological processes, in particular, malignant transformation. The accumulated pool of data on the role of EVs in carcinogenesis has stimulated investigations of the EV-derived cancer markers. The most important factor limiting development of this scientific direction is lack of "gold standards" both for methods of EV isolation from biological fluids and for analyzing their molecular content, including composition of miRNAs. Here we first examined efficacy of various methods for small RNA isolation from EVs contained in ascitic fluid for subsequent miRNA analysis. Comparison of different commercial kits showed advantages of the methods based on phenol-chloroform extraction: Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit. Analysis of the small RNA transcriptome showed presence of various classes of molecules in the EVs, among which proportion of miRNAs averaged 6% and reaching 10% with the Total Exosome RNA & Protein Isolation Kit. The PureLink miRNA Isolation Kit demonstrated the lowest efficiency. The miRNeasy Advanced Serum/Plasma Kit showed the highest concentration of the small RNA fraction, miRNA proportion of which, however, did not exceed that obtained with the miRNeasy Serum/Plasma Kit and Total Exosome RNA & Protein Isolation Kit. Moreover, RT-PCR analysis of the individual molecules showed lower levels of each of investigated miRNAs (miR-1246, miR-200b-5p, miR-200c-3p, and miR-23a-3p) when using the miRNeasy Advanced Serum/Plasma Kit. In conclusion, Total Exosome RNA & Protein Isolation Kit and miRNeasy Serum/Plasma Kit can be considered as optimal kits in terms of performance based on combination of the studied characteristics, including small RNA concentration, percentage of microRNA according to bioanalyzer and sequencing results, and levels of individual miRNAs detected by RT-PCR.

Extracellular Vesicles from Uterine Aspirates Represent a Promising Source for Screening Markers of Gynecologic Cancers.

Extracellular vesicles (EVs), including exosomes, are key factors of intercellular communication, performing both local and distant transfers of bioactive molecules. The increasingly obvious role of EVs in carcinogenesis, similarity of molecular signatures with parental cells, precise selection and high stability of cargo molecules make exosomes a promising source of liquid biopsy markers for cancer diagnosis. The uterine cavity fluid, unlike blood, urine and other body fluids commonly used to study EVs, is of local origin and therefore enriched in EVs secreted by cells of the female reproductive tract. Here, we show that EVs, including those corresponding to exosomes, could be isolated from individual samples of uterine aspirates (UA) obtained from epithelial ovarian cancer (EOC) patients and healthy donors using the ultracentrifugation technique. First, the conducted profiling of small RNAs (small RNA-seq) from UA-derived EVs demonstrated the presence of non-coding RNA molecules belonging to various classes. The analysis of the miRNA content in EVs from UA performed on a pilot sample revealed significant differences in the expression levels of a number of miRNAs in EVs obtained from EOC patients compared to healthy individuals. The results open up prospects for using UA-derived EVs as a source of markers for the diagnostics of gynecological cancers, including EOC.

Detection of Human Papillomavirus Prevalence in Ovarian Cancer by Different Test Systems.

BACKGROUND/AIMS: High-risk human papillomavirus (HPV) infection is associated with different malignancies, but its role in the pathogenesis of ovarian cancer remains inconclusive. Published studies demonstrated a wide variation (0-50%) in HPV prevalence in ovarian cancer. To evaluate the contribution of detection tests to controversial results in different populations, we determined the presence of HPV DNA in Russian ovarian cancer patients using 10 different PCR-based tests. METHODS: Epithelial ovarian adenocarcinomas were tested with 5 general primer sets commonly used for HPV screening of cervical and ovarian cancer and 5 HPV type-specific primers. RESULTS: The use of a single PCR primer set resulted in a wide variation (0-29%) and an underestimation of the incidence of HPV-positive cancers. The combination of MY09/MY11 and GP5+/6+ primers in nested PCR revealed HPV DNA in 53% (18/34) of adenocarcinomas. HPV16 was found in 94% of the HPV-positive cases. In 6/6 positive cases, the active status of HPV16 was demonstrated by RT-PCR detection of E6 and E7 oncogene mRNAs. CONCLUSION: These findings indicate the need to employ multiple PCR-based tests to detect all HPV-positive patients. The identification of viral DNA and oncogene transcripts in cancerous tissues indicate the possible role of HPV in ovarian carcinogenesis in Russia.