Therapy for pancreatic ductal adenocarcinoma remains challenging, and the chances of a complete cure are very limited. As in other types of cancer, the expression and role of miRNAs in controlling the biological properties of this type of tumor have been extensively studied. A better insight into miRNA biology seems critical to refining diagnostics and improving their therapeutic potential. In this study, we focused on the expression of miR-21, -96, -196a, -210, and -217 in normal fibroblasts, cancer-associated fibroblasts prepared from a ductal adenocarcinoma of the pancreas, and pancreatic carcinoma cell lines. We compared these data with miRNAs in homogenates of paraffin-embedded sections from normal pancreatic tissues. In cancer-associated fibroblasts and cancer cell lines, miRNAs differed significantly from the normal tissue. In detail, miR-21 and -210 were significantly upregulated, while miR-217 was downregulated. Similar transcription profiles were earlier reported in cancer-associated fibroblasts exposed to hypoxia. However, the cells in our study were cultured under normoxic conditions. We also noted a relation to IL-6 production. In conclusion, cultured cancer-associated fibroblasts and carcinoma cells reflect miR-21 and -210 expression similarly to the cancer tissue samples harvested from the patients.

• Klíčová slova
• IL-6, cancer-associated fibroblast, hypoxia, miR-21, miR-210, miRNA, pancreas,
• MeSH
• diabetes mellitus MeSH
• duktální karcinom pankreatu * patologie   MeSH
• faciální stigmatizace MeSH
• fibroblasty asociované s nádorem * metabolismus   MeSH
• lidé MeSH
• mikro RNA * genetika   MeSH
• mozeček abnormality   MeSH
• nádorové buněčné linie MeSH
• nádory slinivky břišní * patologie   MeSH
• regulace genové exprese u nádorů MeSH
• růstová retardace plodu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikro RNA * MeSH
• MIRN217 microRNA, human MeSH Prohlížeč

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. They are suggested to be involved in most biological processes of the cell primarily by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Their binding to their target sites is mediated by the Argonaute (AGO) family of proteins. Thus, miRNA target prediction is pivotal for research and clinical applications. Moreover, transfer-RNA-derived fragments (tRFs) and other types of small RNAs have been found to be potent regulators of Ago-mediated gene expression. Their role in mRNA regulation is still to be fully elucidated, and advancements in the computational prediction of their targets are in their infancy. To shed light on these complex RNA-RNA interactions, the availability of good quality high-throughput data and reliable computational methods is of utmost importance. Even though the arsenal of computational approaches in the field has been enriched in the last decade, there is still a degree of discrepancy between the results they yield. This review offers an overview of the relevant advancements in the field of bioinformatics and machine learning and summarizes the key strategies utilized for small RNA target prediction. Furthermore, we report the recent development of high-throughput sequencing technologies, and explore the role of non-miRNA AGO driver sequences.

• Klíčová slova
• computational biology, high-throughput sequencing, machine learning, miRNA target prediction, small RNA target prediction,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant-fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5' RLM-RACE approach.

• Klíčová slova
• RNA interference, Verticillium nonalfalfae, fungal pathogen, microRNA-like RNAs, plant-pathogen interactions,
• MeSH
• Ascomycota genetika   MeSH
• fungální RNA * MeSH
• fylogeneze MeSH
• genová ontologie MeSH
• interakce hostitele a patogenu MeSH
• konformace nukleové kyseliny MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• malá nekódující RNA genetika   MeSH
• mikro RNA genetika   MeSH
• nemoci rostlin mikrobiologie   MeSH
• regulace genové exprese u hub MeSH
• reprodukovatelnost výsledků MeSH
• stanovení celkové genové exprese MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fungální RNA * MeSH
• malá nekódující RNA MeSH
• mikro RNA MeSH

Small RNAs (sRNAs) are 20-30-nucleotide-long, regulatory, noncoding RNAs that induce silencing of target genes at the transcriptional and posttranscriptional levels. They are key components for cellular functions during plant development, hormone signaling, and stress responses. Generated from the cleavage of double-stranded RNAs (dsRNAs) or RNAs with hairpin structures by Dicer-like proteins (DCLs), they are loaded onto Argonaute (AGO) protein complexes to induce gene silencing of their complementary targets by promoting messenger RNA (mRNA) cleavage or degradation, translation inhibition, DNA methylation, and/or histone modifications. This mechanism of regulating RNA activity, collectively referred to as RNA interference (RNAi), which is an evolutionarily conserved process in eukaryotes. Plant RNAi pathways play a fundamental role in plant immunity against viruses and have been exploited via genetic engineering to control disease. Plant viruses of RNA origin that contain double-stranded RNA are targeted by the RNA-silencing machinery to produce virus-derived small RNAs (vsRNAs). Some vsRNAs serve as an effector to repress host immunity by capturing host RNAi pathways. High-throughput sequencing (HTS) strategies have been used to identify endogenous sRNA profiles, the "sRNAome", and analyze expression in various perennial plants. Therefore, the review examines the current knowledge of sRNAs in perennial plants and fruits, describes the development and implementation of RNA interference (RNAi) in providing resistance against economically important viruses, and explores sRNA targets that are important in regulating a variety of biological processes.

• Klíčová slova
• next generation sequencing, perennial plants, resistance, sRNA, viruses,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The oocyte-to-embryo transition (OET) arguably initiates with formation of a primordial follicle and culminates with reprogramming of gene expression during the course of zygotic genome activation. This transition results in converting a highly differentiated cell, i.e. oocyte, to undifferentiated cells, i.e. initial blastomeres of a preimplantation embryo. A plethora of changes occur during the OET and include, but are not limited to, changes in transcription, chromatin structure, and protein synthesis; accumulation of macromolecules and organelles that will comprise the oocyte's maternal contribution to the early embryo; sequential acquisition of meiotic and developmental competence to name but a few. This review will focus on transcriptional and post-transcriptional changes that occur during OET in mouse because such changes are likely the major driving force for OET. We often take a historical and personal perspective, and highlight how advances in experimental methods often catalyzed conceptual advances in understanding the molecular bases for OET. We also point out questions that remain open and therefore represent topics of interest for future investigation.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• embryonální vývoj fyziologie   MeSH
• genom MeSH
• myši MeSH
• oocyty fyziologie   MeSH
• ovariální folikul fyziologie   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells--expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

• MeSH
• buňky 3T3 MeSH
• dvouvláknová RNA metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• imunoprecipitace MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• myši MeSH
• plazmidy genetika   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• průtoková cytometrie MeSH
• regulace genové exprese genetika   MeSH
• reportérové geny genetika   MeSH
• transfekce metody   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dvouvláknová RNA MeSH
• malá interferující RNA MeSH
• protein-serin-threoninkinasy MeSH

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3 % of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P < 0.003)). Increased expression levels of miR-210 in RCC tumor tissue were independently validated. We observed decreased viability of ACHN and CAKI-2 cells and accumulation of CAKI-2 in G2 phase of cell cycle after silencing of miR-210 expression. Downregulation of miR-210 also reduced the migratory and invasive potential of ACHN metastatic RCC cells. Moreover, we showed downregulation of HIF1a protein in both cell lines after miR-210 silencing indicating participation of miR-210 in hypoxic processes of RCC not only through regulation of its target mRNAs but also by indirect regulation of HIF1a. To our knowledge, this is the first report to show miR-210 regulatory effects on cell migration, invasive potential, and HIF1a protein in RCC cells.

• MeSH
• apoptóza MeSH
• buněčný cyklus MeSH
• dospělí MeSH
• down regulace MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• invazivní růst nádoru MeSH
• karcinom z renálních buněk genetika   metabolismus   patologie   MeSH
• kontrolní body buněčného cyklu MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA biosyntéza   genetika   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory ledvin genetika   metabolismus   patologie   MeSH
• pohyb buněk MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• umlčování genů MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktor 1 indukovatelný hypoxií - podjednotka alfa MeSH
• HIF1A protein, human MeSH Prohlížeč
• messenger RNA MeSH
• mikro RNA MeSH
• MIRN210 microRNA, human MeSH Prohlížeč
• nádorové biomarkery MeSH

Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3'-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.

• MeSH
• adenosin metabolismus   MeSH
• buněčné linie MeSH
• deaminace MeSH
• dvouvláknová RNA metabolismus   MeSH
• editace RNA MeSH
• geny mos MeSH
• interferony metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• RNA interference * MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• dvouvláknová RNA MeSH
• interferony MeSH