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There is an urgent need for development of rapid and inexpensive techniques for detection of microRNAs (miRNAs), which are potential biomarkers of various types of cancer. In this paper, we describe a multiplexed electrochemical platform for determination of three cancer‐relevant miRNAs: miR‐21, let‐7a and miR‐31. The strategy combines the use of magnetic beads (MBs) modified with a commercial antibody for the efficient capture of the heteroduplexes formed by hybridization of the target miRNA with DNA probe. Free non‐hybridized region of the DNA probe was thereafter hybridized with two biotin‐labeled auxiliary DNA probes in a process of hybridization chain reaction (HCR), resulting in a long hybrid bearing a large number of biotin molecules. Labeling of these multiple biotin units with streptavidin‐peroxidase conjugates allowed an amplification of the amperometric signal measured after capturing the modified MBs at a screen‐printed carbon electrode array of eight electrodes. The combined strategy demonstrated in a similar assay time significantly higher sensitivity than those previously described using modified MBs with the same capture antibody (without amplification by HCR) or a HCR strategy implemented on the surface of MBs, respectively. The methodology exhibits a good selectivity for discriminating single mismatches and was applied to the determination of the three target miRNAs in total RNA (RNAt) extracted from various cancer cell lines and from cervical precancerous lesions.

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

Regionální centrum aplikované molekulární onkologie, Masarykův onkologický ústav, Brno Souhrn Východiska: I mikroRNA (miRNA) jsou krátké nekódující molekuly RNA regulující genovou expresi vazbou na mRNA. Ovlivňují řadu fyziologických procesů, vč. buněčné proliferace, diferenciace nebo apoptózy, a mají tak výrazný vliv na vývoj nádorových i jiných onemocnění. Představují proto slibnou skupinu nádorových bio markerů využitelných např. v časné dia gnostice, v predikci odpovědi na léčbu, při záchytu relapsu nebo při klasifikaci nádorů do molekulárních subtypů. Cíl: Detekce miRNA vyžaduje různé sofistikované strategie zejména kvůli jejich krátké délce, značné sekvenční podobnosti mezi miRNA patřící do stejné rodiny, i pro jejich často velmi nízkou hladinu ve studovaném vzorku. V této práci jsou zmíněné standardně používané metody, např. reverzní transkripce s následnou polymerázovou řetězovou reakcí, microarrays nebo sekvenování nové generace, a rovněž jsou porovnány různé komerčně dostupné sady pro detekci miRNA. Hlavní důraz je kladen na nově vyvíjené technologie a metody, které by mohly současnou analýzu zlevnit nebo urychlit. Představeny jsou např. alternativní amplifikační techniky (izotermální amplifikace, hybridizační řetězová reakce), různé typy nanomateriálů, speciální proteiny se schopností vázat miRNA, a řada bio senzorů využívající optickou nebo elektrochemickou detekci. Závěr: Důležitost miRNA vedla k obrovskému nárůstu počtu nově vyvíjených metod. Většina z nich ovšem nebyla testována na klinickém materiálu, takže je těžké určit jejich eventuální aplikovatelnost do praxe. Pro komerční využití nových metod bude proto nutné provést jejich přísnou validaci pomocí standardních metod nejenom na modelových systémech, ale zejména u klinických vzorků. Klíčová slova mikroRNA-regulace genové exprese-nádorové bio markery-RT-PCR-bio senzory Summary Background: MicroRNA (miRNA) are a class of short non-coding RNA molecules that regulate gene expression at the post-transcription level by binding to mRNA. By affecting many physiological processes, including cellular proliferation, differentiation, and apoptosis, they have a major impact on the development of cancer as well as other diseases. Hence, miRNAs could serve as potential tumor biomarkers in e.g. early diagnostics, predicting responses to therapy, monitoring relapse, and molecular classification of tumors. Aim: miRNA detection requires various sophisticated strategies due to the small size, sequence similarity among family members, and often very low levels of miRNAs in analyzed samples. This review describes standard techniques of miRNA detection, such as the reverse transcriptase polymerase chain reaction, microarrays, and next-generation sequencing, and compares several commercially available detection kits. Major emphasis is given to newly developed technologies and methods, which could make the analysis cheaper and quicker. We present, for instance, alternative amplification techniques (isothermal amplification and the hybridization chain reaction), differ...