INTRODUCTION: Cervical cancer causes approximately 350,000 deaths each year. The availability of sensitive and specific diagnostic tests to detect cervical cancer in its early stages is essential to improve survival rates. METHODS: In this study, we compared two strategies for selecting endogenous controls: miRNA profiling by small-RNA sequencing and a commercially available microfluidic card with 30 recommended endogenous controls preloaded by the manufacturer. We used the RefFinder algorithm and coefficient of variation to select endogenous controls. We selected the combination of miR-181a-5p and miR-423-3p as the most optimal normalizer. In the second part of this study, we determined the differential expression (between tumor/non-tumor groups) of microRNA in cervical cancer FFPE tissue samples. We determined the comprehensive miRNA expression profile using small-RNA sequencing technology and verified the results by real-time PCR. We determined the relative expression of selected miRNAs using the 2-ΔΔCt method. RESULTS: We detected statistically significant upregulation of miR-320a-3p, miR-7704, and downregulation of miR-26a-5p in the tumor group compared to the control group. The combination of these miRNAs may have the potential to be utilized as a diagnostic panel for cervical cancer. Using ROC curve analysis, the proposed panel showed 93.33% specificity and 96.97% sensitivity with AUC = 0.985. CONCLUSIONS: We proposed a combination of miR-181a-5p and miR-423-3p as optimal endogenous control and detected potentially significant miRNAs (miR-320a-3p, miR-7704, miR-26a-5p). After further validation of our results, these miRNAs could be used in a diagnostic panel for cervical cancer.

• Klíčová slova
• NGS, cervical cancer, diagnostic biomarkers, endogenous control, microRNA, normalization strategy,
• Publikační typ
• časopisecké články MeSH

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol's performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

• Klíčová slova
• diagnostics, miRNA, small RNA-seq,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Adenoid cystic carcinoma (ACC) is among the most frequent malignancies of the salivary gland, and is notorious for its prolonged clinical course characterized by frequent recurrences often years after initial treatment. No molecular marker has been shown to have independent prognostic value in ACC, including characteristic gene fusions involving MYB, MYBL1, and NFIB. MicroRNA has been shown to be associated with clinical outcome in numerous malignancies, including one study of ACC, warranting further validation of this class of markers in this disease. Here, we investigate the prognostic value of microRNA in two ACC cohorts: a training cohort (n = 64) and a validation cohort (n = 120) with microarray and qPCR. In the training cohort, multivariate analysis of microarray data found high expression of hsa-miR-6835-3p to be associated with reduced recurrence-free survival (RFS) (p = 0.016). Measuring the highest ranking microRNAs identified in survival analysis in the same cohort, qPCR identified high expression of hsa-miR-4676 to be associated with reduced overall survival (OS) and high expression of hsa-mir-1180 to be associated with improved RFS. This was not confirmed in the validation cohort, in which qPCR identified high expression of hsa-mir-21, hsa-mir-181a-2, and hsa-mir-152 to be associated with reduced OS and high expression of hsa-miR-374c to be associated with improved RFS. Interestingly, two distinct subsets of ACC separated in microRNA expression irrespective of gene fusion status, but without significant difference in outcome. Collectively, qPCR identified several microRNAs associated with OS and RFS, and different subsets of ACC separated according to microRNA expression, suggestive of ACC being a heterogeneous group of malignancies in its microRNA profile.

• Klíčová slova
• Adenoid cystic carcinoma, MYB, MYBL1, Prognosis, Salivary gland, microRNA,
• MeSH
• adenoidně cystický karcinom genetika   mortalita   patologie   MeSH
• dospělí MeSH
• fúze genů * MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA analýza   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádory slinných žláz genetika   mortalita   patologie   MeSH
• prognóza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikro RNA MeSH

Growing evidence suggests that microRNAs are involved in the development and progression of colorectal cancer (CRC). In the present study, deregulation and functioning of tumor-suppressive miR-215-5p was evaluated in CRC. In total, 448 tumor tissues and 325 paired adjacent healthy tissues collected from Czech and Spain cohorts of CRC patients have been used for miR-215-5p expression analyses. A series of in vitro experiments have been performed using transient transfection of miR-215-5p mimics into four CRC cell lines to identify specific cellular processes affected by miR-215-5p. Further, the effects of miR-215-5p on tumor growth were evaluated in vivo using NSG mice and stable cell line overexpressing miR-215-5p. Target mRNAs of miR-215-5p were tested using luciferase assay and western blot analyses. We found that miR-215-5p is significantly downregulated in tumor tissues compared with non-tumor adjacent tissues and its decreased levels correlate with the presence of lymph node metastases, tumor stage, and shorter overall survival in CRC patients. Overexpression of miR-215-5p significantly reduced proliferation, clonogenicity, and migration of CRC cells, lead to cell cycle arrest in G2/M phase and p53-dependent induction of apoptosis. The ability of miR-215-5p to inhibit tumor growth was confirmed in vivo. Finally, we confirmed epiregulin and HOXB9 to be the direct targets of miR-215-5p. As epiregulin is EGFR ligand and HOXB9 is its transcriptional inducer, we suggest that the main molecular link between miR-215-5p and CRC cells phenotypes presents the EGFR signaling pathway, which is one of the canonical pathogenic pathways in CRC.

• Publikační typ
• časopisecké články MeSH

AIMS: The distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. METHODS: A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. RESULTS: Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). CONCLUSIONS: A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

• Klíčová slova
• DIAGNOSTICS, LABORATORY TESTS, MOLECULAR ONCOLOGY, THYROID, THYROID CANCER,
• MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA metabolismus   MeSH
• nádory štítné žlázy diagnóza   MeSH
• odchylka pozorovatele MeSH
• prediktivní hodnota testů MeSH
• tenkojehlová biopsie MeSH
• uzly štítné žlázy diagnóza   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• validační studie MeSH
• Názvy látek
• mikro RNA MeSH

Purpose: Characterization of colorectal cancer transcriptome by high-throughput techniques has enabled the discovery of several differentially expressed genes involving previously unreported miRNA abnormalities. Here, we followed a systematic approach on a global scale to identify miRNAs as clinical outcome predictors and further validated them in the clinical and experimental setting.Experimental Design: Genome-wide miRNA sequencing data of 228 colorectal cancer patients from The Cancer Genome Atlas dataset were analyzed as a screening cohort to identify miRNAs significantly associated with survival according to stringent prespecified criteria. A panel of six miRNAs was further validated for their prognostic utility in a large independent validation cohort (n = 332). In situ hybridization and functional experiments in a panel of colorectal cancer cell lines and xenografts further clarified the role of clinical relevant miRNAs.Results: Six miRNAs (miR-92b-3p, miR-188-3p, miR-221-5p, miR-331-3p, miR-425-3p, and miR-497-5p) were identified as strong predictors of survival in the screening cohort. High miR-188-3p expression proves to be an independent prognostic factor [screening cohort: HR = 4.137; 95% confidence interval (CI), 1.568-10.917; P = 0.004; validation cohort: HR = 1.538; 95% CI, 1.107-2.137; P = 0.010, respectively]. Forced miR-188-3p expression increased migratory behavior of colorectal cancer cells in vitro and metastases formation in vivo (P < 0.05). The promigratory role of miR-188-3p is mediated by direct interaction with MLLT4, a novel identified player involved in colorectal cancer cell migration.Conclusions: miR-188-3p is a novel independent prognostic factor in colorectal cancer patients, which can be partly explained by its effect on MLLT4 expression and migration of cancer cells. Clin Cancer Res; 23(5); 1323-33. ©2016 AACR.

• MeSH
• genom lidský MeSH
• HCT116 buňky MeSH
• karcinogeneze genetika   MeSH
• kineziny genetika   MeSH
• kolorektální nádory genetika   patologie   MeSH
• lidé MeSH
• mikro RNA genetika   izolace a purifikace   MeSH
• myosiny genetika   MeSH
• myši MeSH
• nádorové biomarkery genetika   MeSH
• prognóza MeSH
• transkriptom genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• AFDN protein, human MeSH Prohlížeč
• kineziny MeSH
• mikro RNA MeSH
• MIRN188 microRNA, human MeSH Prohlížeč
• myosiny MeSH
• nádorové biomarkery MeSH

BACKGROUND: The majority of thyroid nodules are diagnosed using fine-needle aspiration (FNA) biopsies. The authors recently described the clinical validation of a molecular microRNA-based assay, RosettaGX Reveal, which can diagnose thyroid nodules as benign or suspicious using a single stained FNA smear. This paper describes the analytical validation of the assay. METHODS: More than 800 FNA slides were tested, including slides stained with Romanowsky-type and Papanicolaou stains. The assay was examined for the following features: intranodule concordance, effect of stain type, minimal acceptable RNA amounts, performance on low numbers of thyroid cells, effect of time since sampling, and analytical sensitivity, specificity, and reproducibility. RESULTS: The assay can be run on FNA slides for which as little as 1% of the cells are thyroid epithelial cells or from which only 5 ng of RNA have been extracted. Samples composed entirely of blood failed quality control and were not classified. Stain type did not affect performance. All slides were stored at room temperature. However, the length of time between FNA sampling and processing did not affect assay performance. There was a high level of concordance between laboratories (96%), and the concordance for slides created from the same FNA pass was 93%. CONCLUSIONS: The microRNA-based assay was robust to various physical processing conditions and to differing sample characteristics. Given the assay's performance, robustness, and use of routinely prepared FNA slides, it has the potential to provide valuable aid for physicians in the diagnosis of thyroid nodules. Cancer Cytopathol 2016;124:711-21. © 2016 Rosetta Genomics. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

• Klíčová slova
• fine-needle aspiration (FNA), indeterminate, microRNA, molecular test, nodules, smears, thyroid,
• MeSH
• cytodiagnostika metody   MeSH
• diagnostické techniky molekulární metody   normy   MeSH
• folikulární papilární karcinom diagnóza   genetika   MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• nádorové biomarkery genetika   MeSH
• nádory štítné žlázy diagnóza   genetika   MeSH
• odběr biologického vzorku metody   MeSH
• prognóza MeSH
• reprodukovatelnost výsledků MeSH
• tenkojehlová biopsie MeSH
• uzly štítné žlázy diagnóza   genetika   MeSH
• validační studie jako téma MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikro RNA MeSH
• nádorové biomarkery MeSH

Gastrointestinal cancer accounts for the 20 most frequent cancer diseases worldwide and there is a constant urge to bring new therapeutics with new mechanism of action into the clinical practice. Quantity of in vitro and in vivo evidences indicate, that exogenous change in pathologically imbalanced microRNAs (miRNAs) is capable of transforming the cancer cell phenotype. This review analyzed preclinical miRNA-based therapy attempts in animal models of gastric, pancreatic, gallbladder, and colorectal cancer. From more than 400 original articles, 26 was found to assess the effect of miRNA mimics, precursors, expression vectors, or inhibitors administered locally or systemically being an approach with relatively high translational potential. We have focused on mapping available information on animal model used (animal strain, cell line, xenograft method), pharmacological aspects (oligonucleotide chemistry, delivery system, posology, route of administration) and toxicology assessments. We also summarize findings in the field pharmacokinetics and toxicity of miRNA-based therapy.

• Klíčová slova
• animal model, colorectal cancer, gallbladder cancer, gastric cancer, mice, microRNA, pancreatic cancer, preclinical testing,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The most important reason for therapy failure in pediatric acute myeloid leukemia (AML) is relapse. In order to identify miRNAs that contribute to the clonal evolution towards relapse in pediatric AML, miRNA expression profiling of 127 de novo pediatric AML cases were used. In the diagnostic phase, no miRNA signatures could be identified that were predictive for relapse occurrence, in a large pediatric cohort, nor in a nested mixed lineage leukemia (MLL)-rearranged pediatric cohort. AML with MLL- rearrangements are found in 15-20% of all pediatric AML samples, and reveal a relapse rate up to 50% for certain translocation partner subgroups. Therefore, microRNA expression profiling of six paired initial diagnosis-relapse MLL-rearranged pediatric AML samples (test cohort) and additional eight paired initial diagnosis-relapse samples with MLL-rearrangements (validation cohort) was performed. A list of 53 differentially expressed miRNAs was identified of which the miR-106b~25 cluster, located in intron 13 of MCM7, was the most prominent. These differentially expressed miRNAs however could not predict a relapse in de novo AML samples with MLL-rearrangements at diagnosis. Furthermore, higher mRNA expression of both MCM7 and its upstream regulator E2F1 was found in relapse samples with MLL-rearrangements. In conclusion, we identified the miR-106b~25 cluster to be upregulated in relapse pediatric AML with MLL-rearrangements.

• Klíčová slova
• MLL, acute myeloid leukemia, miR-106b~25, relapse,
• MeSH
• akutní myeloidní leukemie genetika   patologie   MeSH
• dítě MeSH
• histonlysin-N-methyltransferasa genetika   metabolismus   MeSH
• introny genetika   MeSH
• karcinogeneze genetika   MeSH
• klonální evoluce MeSH
• kohortové studie MeSH
• kojenec MeSH
• lidé MeSH
• lokální recidiva nádoru genetika   patologie   MeSH
• MCM komplex, komponenta 7 metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mikro RNA genetika   MeSH
• mladiství MeSH
• multigenová rodina MeSH
• předškolní dítě MeSH
• protoonkogenní protein MLL genetika   MeSH
• regulace genové exprese u leukemie * MeSH
• stanovení celkové genové exprese MeSH
• transkripční faktor E2F1 metabolismus   MeSH
• translokace genetická MeSH
• upregulace MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• E2F1 protein, human MeSH Prohlížeč
• histonlysin-N-methyltransferasa MeSH
• KMT2A protein, human MeSH Prohlížeč
• MCM komplex, komponenta 7 MeSH
• MCM7 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• mikro RNA MeSH
• MIRN106 microRNA, human MeSH Prohlížeč
• protoonkogenní protein MLL MeSH
• transkripční faktor E2F1 MeSH

BACKGROUND: MicroRNAs (miRs) represent a distinct class of posttranscriptional modulators of gene expression with remarkable stability in sera. Several miRs are oncogenic (oncomiRs) and are deregulated in the pathogenesis of breast cancer and function to inhibit tumor suppressors. Routine blood monitoring of these circulating tumor-derived products could be of significant benefit to the diagnosis and relapse detection of early-stage breast cancer (EBC) patients. METHODS: Aim of this project was to determine expression of miR-155, miR-19a, miR-181b, miR-24, relative to let-7a in sera of 63 patients with EBC and 21 healthy controls. Longitudinal multivariate data analysis was performed to stochastically model the serum levels of each of the oncomiRs during disease phases: from diagnosis, after surgery, and following chemo/radiotherapy. Moreover, this analysis was utilized to evaluate oncomiR levels in EBC patients subgrouped using current clinical prognostic factors including HER2, Ki-67, and grade III. RESULTS: EBC patients significantly over-express the oncomiRs at the time of diagnosis. Following surgical resection the serum levels of miR-155, miR-181b, and miR-24 significantly decreased (p = 1.89e-05, 5.41e-06, and 0.00638, respectively) whereas the miR-19a decreased significantly after the therapy (p = 0.00869). Furthermore, in case of high-risk patients serum levels of miR-155, miR-19a, miR-181b, and miR-24 are significantly more abundant in comparison to low-risk group (p = 0.026, 0.02567, 0.0250, and 0.00990) and show a decreasing trend upon therapy. CONCLUSIONS: OncomiRs are significantly more abundant in the sera of EBC patients compared to controls at diagnosis. Differences in oncomiR levels reflecting EBC risk were also observed. Testing the oncomiRs may be useful for diagnostic purpose and possibly also for relapse detection in follow-up studies of EBC.

• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA krev   genetika   MeSH
• nádorové biomarkery krev   genetika   MeSH
• nádory prsu diagnóza   genetika   terapie   MeSH
• prognóza MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• stupeň nádoru MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• MIRN155 microRNA, human MeSH Prohlížeč
• MIrn181 microRNA, human MeSH Prohlížeč
• MIRN19A microRNA, human MeSH Prohlížeč
• MIRN24 microRNA, human MeSH Prohlížeč
• nádorové biomarkery MeSH