Long noncoding RNAs (lncRNAs) are a relatively well-characterized class of noncoding RNA (ncRNA) molecules, involved in the regulation of various cell processes, including transcription, intracellular trafficking, and chromosome remodeling. Their deregulation has been associated with the development and progression of various cancer types, the fact which makes them suitable as biomarkers for cancer diagnosis and prognosis. In recent years, detection of cancer-associated lncRNAs in body fluids of cancer patients has proven itself as an especially valuable method to effectively diagnose cancer. Cancer diagnosis and prognosis employing circulating lncRNAs are preferential when compared to classical biopsies of tumor tissues, especially due to their noninvasiveness, and have great potential for routine usage in clinical practice. Thus, this review focuses on summarizing the perspectives of lncRNAs as biomarkers in cancer, based on evaluating their expression profiles determined in body fluids of cancer patients.

Circular RNAs (circRNAs) are a class of noncoding RNAs (ncRNAs) that form covalently closed continuous loop structures, lacking the terminal 5′ and 3′ ends. CircRNAs are generated in the process of back-splicing and can originate from different genomic regions. Their unique circular structure makes circRNAs more stable than linear RNAs. In addition, they also display insensitivity to ribonuclease activity. Generally, circRNAs function as microRNA (miRNA) sponges and have a regulatory role in transcription and translation. They may be also translated in a cap-independent manner in vivo, to generate specific proteins. In the last decade, next-generation sequencing techniques, especially RNA-seq, have revealed great abundance and also dysregulation of many circRNAs in various diseases, suggesting their involvement in disease development and progression. Regarding their high stability and relatively specific differential expression patterns in tissues and extracellular environment (e.g., body fluids), they are regarded as promising novel biomarkers in cancer. Therefore, we focus this review on describing circRNA biogenesis, function, and involvement in human cancer development and address the potential of circRNAs to be effectively used as novel cancer diagnostic and prognostic biomarkers.

Objectives To identify dysregulated microRNAs (miRNAs) and their gene targets in temporal arteries from GCA patients, and determine their association with GCA pathogenesis and related arterial wall remodelling. Methods We included 93 formalin-fixed, paraffin-embedded temporal artery biopsies (TABs) from treatment-naïve patients: 54 positive and 17 negative TABs from clinically proven GCA patients, and 22 negative TABs from non-GCA patients. miRNA expression analysis was performed with miRCURY LNA miRNome Human PCR Panels and quantitative real-time PCR. miRNA target gene prediction and pathway enrichment analysis was performed using the miRDB and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases, respectively. Results Dysregulation of 356 miRNAs was determined in TAB-positive GCA arteries, among which 78 were significantly under-expressed and 22 significantly overexpressed above 2-fold, when compared with non-GCA controls. Specifically, TAB-positive GCA arteries were characterized by a significant overexpression of ‘pro-synthetic’ (miR-21-3p/-21-5p/-146a-5p/-146b-5p/-424-5p) and under-expression of ‘pro-contractile’ (miR-23b-3p/-125a-5p/-143-3p/-143-5p/-145-3p/-145-5p/-195-5p/-365a-3p) vascular smooth muscle cell phenotype-associated regulatory miRNAs. These miRNAs targeted gene pathways involved in the arterial remodelling and regulation of the immune system, and their expression correlated with the extent of intimal hyperplasia in TABs from GCA patients. Notably, the expression of miR-21-3p/-21-5p/-146a-5p/-146b-5p/-365a-3p differentiated between TAB-negative GCA arteries and non-GCA temporal arteries, revealing these miRNAs as potential biomarkers of GCA. Conclusion Identification of dysregulated miRNAs involved in the regulation of the vascular smooth muscle cell phenotype and intimal hyperplasia in GCA arterial lesions, and detection of their expression profiles, enables a novel insight into the complexity of GCA pathogenesis and implies their potential utilization as diagnostic and prognostic biomarkers of GCA.

IgA vasculitis (IgAV) represents a common systemic vasculitis in pediatric and adult population. Our current knowledge of disease pathogenesis is still very limited, without information on miRNAs in IgAV. The aim of our study was to determine the expression of five pre-selected miRNAs (miRNA-146a-5p, miRNA-148-3p, miRNA-155-5p, miRNA-223-3p, and let-7b) in the affected skin of adult IgAV patients. The study included 65 skin samples from consecutive, untreated IgAV patients (61.5% male, median age 67.6 years, range 29-91), diagnosed between October 2014 and September 2016, and 20 samples of normal skin from healthy volunteers. Total RNA was isolated from tissue sections of formalin-fixed, paraffin-embedded samples. Expression of miRNAs was measured using qRT-PCR. To present relative miRNA expression, the ΔΔCT method was used. Skin miRNA expression was correlated to clinical characteristics of adult IgAV patients. We found significantly higher levels of miRNA-155-5p, miRNA-223-3p, and let-7b in the affected skin compared to controls (18.6-fold, 6.4-fold, and 7.9-fold higher respectively). Contrary, the miRNA 148-3p expression was significantly lower (2.2-fold). The expression of the miRNA-146-5p showed near normal levels. Patients with necrotic skin lesions had significantly higher miRNA-223 tissue expression than those with non-necrotic purpura (p = 0.029). Gastrointestinal tract involvement inversely correlated with the expression of miRNA-155-5p and/or miRNA-146a-5p in affected skin. Altered expression of miRNA-148b-3p, miRNA-155-5p, miRNA-223-3p, and let-7b was found in vasculitic skin lesions in IgAV. Additionally, we found a positive association between the severity of purpura and skin miRNA-223-3p expression. Aberrantly expressed miRNAs could represent a biomarker in adult IgAV.

ObjectivesThe aim of this study was to quantitatively assess distinct immune cell subsets comprising inflammatory infiltrate in temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA), and to link the obtained histopathological data with expression profiles of immune-related microRNAs (miRNAs).MethodsThe study included 68 formalin-fixed, paraffin-embedded TABs from treatment-naïve patients, including 30 histologically positive GCA and 16 negative GCA TABs, and 22 control non-GCA TABs. Quantitative assessment of histological parameters was performed using histopathological and immunohistochemical techniques. miRNA expression analysis was performed by quantitative real-time PCR.ResultsIntense transmural mononuclear inflammatory infiltrates in TAB-positive GCA arteries were predominantly composed of CD3+, CD4+ and CD8+ T lymphocytes, and CD68+ macrophages, accompanied by a strong nuclear overexpression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATC) in the lymphocyte infiltrate fraction. Furthermore, TAB-positive GCA arteries were characterized by significant overexpression of nine pro-inflammatory miRNAs (miR-132-3p/-142-3p/-142-5p/-155-5p/-210-3p/-212-3p/-326/-342-5p/-511-5p) and a significant under-expression of six regulatory immune-related miRNAs (miR-30a-5p/-30b-5p/-30c-5p/-30d-5p/-30e-5p/-124-3p), whose expression levels significantly associated with most evaluated histopathological parameters. Notably, we revealed miR-132-3p/-142-3p/-142-5p/-155-5p/-212-3p/-511-5p as major promoters of arterial inflammation and miR-30a-5p/-30c-5p/-30d-5p as putative regulators of NFATC signaling in TAB-positive GCA arteries.ConclusionOverall, we demonstrated that an altered arterial tissue-specific pro-inflammatory miRNA signature favors enhanced T cell-driven inflammation and macrophage activity in TAB-positive GCA arteries. Moreover, dysregulation of several immune-related miRNAs seems to contribute crucially to GCA pathogenesis, through impairing their regulatory activity towards T cell-mediated immune responses driven by the calcineurin (CaN)/NFAT signaling pathway, indicating their therapeutic, diagnostic and prognostic potential.

Mycoplasma synoviae and Newcastle disease virus (NDV) are 2 avian pathogens that cause modulation in expression of a variety of cytokine and chemokine genes in chickens. However, there is limited data about gene modulation after coinfection with these 2 pathogens and even less data about gene modulation after infection of chicken embryos. In this study, the effect of M. synoviae type strain WVU 1853 and lentogenic LaSota vaccine strain of NDV infection on cytokine and chemokine gene expression in chicken embryos was analyzed in the liver, spleen, bursa of Fabricius, and thymus by using quantitative real-time PCR. Three types of infection were performed; infection with M. synoviae on d 10, infection with NDV on d 17; and consecutive infection with both pathogens, where M. synoviae was inoculated on d 10 and NDV on d 17. Thus, simulation of consecutive infection that may occur after NDV infection of the M. synoviae-infected host was performed. Mycoplasma synoviae infection of embryos resulted in intensive upregulation of cytokine and chemokine genes, including interferon (IFN)-γ, IL-1β, IL-6, IL-12p40, IL-16, IL-18, MIP-1β (CCL4), inducible nitric oxide synthase (iNOS), XCL1, and lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF), with different expression profiles in the 4 organs. Inoculation of lentogenic NDV significantly upregulated IFN-γ, IL-6, and IL-16 genes in spleen and IFN-γ, IL-1β, IL-2, IL-16, IL-21, XCL1, and MIP-1β (CCL4) genes in the thymus, but to a lesser extent than M. synoviae. However, no genes were upregulated by NDV in the liver and bursa of Fabricius. Overall effect of NDV inoculation, regarding the number of modulated cytokine and chemokine genes and the extent of expression, was lower than M. synoviae. When NDV was introduced after on-going M. synoviae infection, most M. synoviae-induced cytokine and chemokine genes were significantly downregulated. This study provides the first evidence in chicken embryos that consecutive infection with NDV could suppress expression of cytokine and chemokine genes being significantly upregulated by the previous M. synoviae infection.

and rapid quantification results (Pfaffl, 2001; Yuan et al., 2006). Because of the lacking consensus on how to best perform qPCR, MIQE guidelines have been developed to uniform qPCR experiment setup, optimization and data analysis, making the protocols comparable between different research groups and organizations and ensuring the relevance, accuracy, correct interpretation and repeatability of the results (Bustin et al., 2009). When analyzing gene expression, qPCR data can be subjected to absolute or relative quantification (Livak and Schmittgen, 2001; Pfaffl, 2001; Yuan et al., 2006). Absolute quantification employs internal or external cali-Comparison of methods for relative quantification of gene expression using real-time PCR Quantitative real-time PCR (qPCR) has become a widely used tool for quantifying gene expression. Several methods for relative quantification have been developed, enabling rapid and reliable detection and quantification of specific nucleic acids. These methods, based on qPCR include: the standard curve method, the efficiency calibrated method and the 2 −ΔΔCq method. Here we analyzed if these three methods generate comparable results. To evaluate their performance, we analyzed the expression of the nuclease gene MS53_0284 from Mycoplasma synoviae type strain WVU 1853 during in vitro infection of CEC-32 cells, using qPCR. As determined, all three methods generated comparable and reliable results when all necessary conditions were fulfiled. Also, the efficiency calibrated and the standard curve methods were more suitable for quantifying small differences in relative gene expression than the 2 −ΔΔCq method. Key words: molecular genetics / genes / gene expression / quantitative real-time PCR / methods Primerjava metod za relativno kvantifikacijo genskega izražanja s PCR v realnem času Metoda kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) je postala najpogosteje uporabljen način analize izražanja genov. Razvitih je bilo več metod za relativno kvantifikacijo genske ekspresije, ki omogočajo hitro in zanesljivo detekcijo ter kvantifikacijo specifičnih nukleinskih kislin. Mednje spadajo: metoda z umeritveno krivuljo, metoda z upoštevanjem učinkovitosti pomnoževanja in metoda po enačbi 2 −ΔΔCq. V tej študiji smo z analizirajem izražanja gena MS53_0284, ki kodira nukleazo pri bakteriji Mycoplasma synoviae WVU 1853, po okužbi celic CEC-32 in vitro s qPCR preverili primerljivost rezultatov, dobljenih z uporabo omenjenih metod. Pokazali smo, da z upoštevanjem potrebnih pogojev z omenjenimi metodami pridobimo primerljive rezultate ter da sta metoda z umeritveno krivuljo in metoda z upoštevanjem učinkovitosti pomnoževanja primernejši za ugotavljanje majhnih razlik v izražanju genov kot metoda po enačbi 2 −ΔΔCq .

Aims IgA vasculitis (IgAV) is a common small‐vessel systemic vasculitisthat is histologically characterised by granulocyte infiltration and IgA deposition in vessel walls. Information on microRNA (miRNA) involvement inIgAVis limited. The aim of this study was to analyse the association between histopathological changes and expression profiles of 14 miRNAs in the affected skin of 70 adult patients with IgAV. Methods and results miRNA expression analysis was performed by quantitative real‐time polymerase chain reaction and evaluation of histopathological changes by light and immunofluorescence microscopy on formalin‐fixed paraffin‐embedded skin excision samples. In IgAV‐affected skin, granulocyte infiltration was significantly associated with vessel fibrinoid necrosis. Of the analysed miRNAs, four showed two‐fold increased expression (let‐7d, let‐7f, miR‐21‐5p, and miR‐203‐3p), five showed five‐fold increased expression (let‐7b, miR‐17‐5p, miR‐155‐5p, miR‐423‐5p, and miR‐451a), and threeshowed 15‐fold increased expression (let‐7a, miR‐21‐3p, miR‐223‐3p), as compared with controls (all P < 0.001). miR‐146a‐5p and miR‐148b‐3p showed three‐fold decreased expression (P = 0.981 and P < 0.001). The expression of miR‐223‐3p also showed a significant positive association with granulocyte infiltration and fibrinoid necrosis. Conclusions Altered miRNA expression, especially of miRNA‐223‐3p, may be associated with the skin inflammatory state in IgAV. The majority of aberrantly expressed miRNAs in IgAV‐affected skin are known to influence the nuclear factor‐κB signalling pathway, which is crucial for activation of key proinflammatory genes, including those encoding tumour necrosis factor‐α, interleukin (IL)‐6, and IL‐8. Furthermore, miR‐146a‐5p and miR‐148b‐3p, which are negative regulators of inflammatory gene expression, showed decreased expression and could contribute to the exaggerated inflammation. Further investigation of miRNA expression in the affected tissues could improve our knowledge of IgAV pathogenesis, and possibly help to identify novel biomarkers in body fluids.