OBJECTIVES: Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. METHODS: Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. RESULTS: Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. CONCLUSIONS: We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.
- MeSH
- extracelulární vezikuly * genetika metabolismus MeSH
- gelová chromatografie MeSH
- lidé MeSH
- RNA MeSH
- stanovení celkové genové exprese * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Stage II colon cancer (CC) encompasses a heterogeneous group of patients with diverse survival experiences: 87% to 58% 5-year relative survival rates for stages IIA and IIC, respectively. While stage IIA patients are usually spared the adjuvant chemotherapy, some of them relapse and may benefit from it; thus, their timely identification is crucial. Current gene expression signatures did not specifically target this group nor did they find their place in clinical practice. Since processes at invasion front have also been linked to tumor progression, we hypothesize that aside from bulk tumor features, focusing on the invasion front may provide additional clues for this stratification. A retrospective matched case-control collection of 39 stage IIA microsatellite-stable (MSS) untreated CCs was analyzed to identify prognostic gene expression-based signatures. The endpoint was defined as relapse within 5 years vs. no relapse for at least 6 years. From the same tumors, three different classifiers (bulk tumor, invasion front, and constrained baseline on bulk tumor) were developed and their performance estimated. The baseline classifier, while the weakest, was validated in two independent data sets. The best performing signature was based on invasion front profiles [area under the receiver operating curve (AUC) = 0.931 (0.815-1.0)] and contained genes associated with KRAS pathway activation, apical junction complex, and heme metabolism. Its combination with bulk tumor classifier further improved the accuracy of the predictions.
- Publikační typ
- časopisecké články MeSH
Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.
Východiska: MikroRNA (miRNA) jsou jednou ze skupin krátkých nekódujících RNA, které na posttranskripční úrovni regulují genovou expresi. Jsou zapojeny do klíčových buněčných procesů a podílejí se na vzniku a progresi mnohých onemocnění. Hladiny miRNA mohou odrážet fyziologický stav organizmů, a proto jsou v rámci biomarkerových studií analyzovány jejich expresní profily. Díky svým vlastnostem se jeví jako slibné diagnostické, prognostické a prediktivní biomarkery nádorových onemocnění. Nedávné studie poukazují na existenci sekvenčních variant miRNA, tzv. izomiRs, které se od anotovaných miRNA liší pozměněnou sekvencí v důsledku posttranskripčních modifikací. Tyto izomiRs mohou vykazovat vyšší zastoupení než některé kanonické miRNA. Bližší studium izomiRs odhaluje jejich nenáhodnou distribuci i odlišné biologické vlastnosti a poukazuje tak na možný biologický význam modifikací sekvence. Přítomnost izomiRs může také zásadně ovlivňovat výsledky biomarkerových studií. V současné době je pozornost zaměřena na jejich možný klinický význam. Cíl: Cílem této práce je poskytnout přehled současných poznatků o sekvenčních variantách miRNA. V práci jsou popsány mechanizmy vzniku izomiRs a vliv heterogenity v sekvenci na stabilitu, funkci a analýzu miRNA. Prostor je věnován také roli izomiRs v biomarkerových studiích.
Background: MicroRNA (miRNA) are a class of short non-coding RNAs that regulate gene expression at the posttranscriptional level. They are involved in key cellular processes and development as well as progression of many diseases. Their levels reflect the physiological state of organisms; therefore, the expression profiles of these molecules are analyzed in biomarker studies. Due to their properties, miRNA appear to be promising diagnostic, prognostic and predictive biomarkers of cancer. Recent studies indicate the existence of sequence variants in miRNA, so-called isomiRs, which differ from the annotated miRNAs by altered sequences due to posttranscriptional modifications. These isomiRs may have a higher abundance than canonical miRNA. The characterization of isomiRs reveals their regulated distribution and different biological properties and thus suggest the possible biological significance of the modifications. The presence of isomiRs can also significantly affect the results of biomarker studies. Currently, the research is focused on their possible clinical significance. Purpose: The aim of this review is to provide an overview of current knowledge about sequence variants in miRNA. The review summarizes the mechanisms of isomiRs biogenesis and describes the effects of sequence heterogeneity on miRNA stability, function and analysis. Subsequently, the role of isomiRs in biomarker studies is discussed.
