Preservation of 5-Hydroxymethylcytosine Levels in LRIG1 across Genomic DNA and Cell-Free DNA in Glioma Patients.

Cell-free DNA (cfDNA) has recently emerged as a promising minimally invasive diagnostic biomarker for various cancers. In this study, our aim was to identify cfDNA biomarkers by investigating genes that displayed significant differences between glioma patients and their corresponding controls. To accomplish this, we utilized publicly available data from the Gene Expression Omnibus, focusing on 5-hydroxymethylcytosine (5hmC) profiles in both cfDNA and genomic DNA (gDNA) from glioma patients and healthy individuals. The intersection of gene lists derived from these comparative analyses unveiled LRIG1 and ZNF703 as the two genes with elevated 5hmC levels in both the cfDNA of glioma patients and gDNA of glioma tissue compared to their respective controls. The gene expression data revealed both genes were upregulated in glioma tissue compared to normal brain tissue. Integration of 5hmC data revealed a strong positive correlation in the glioma tissue group between 5hmC and the gene expression of the LRIG1 gene. Furthermore, exploration using the AmiCa web tool indicated that LRIG1 gene expression was elevated compared to 17 other cancers included in the database, emphasizing its potential as a distinctive biomarker across multiple cancer types.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Evaluation of Arterial Histopathology and microRNA Expression That Underlie Ultrasonography Findings in Temporal Arteries of Patients with Giant Cell Arteritis.

The aim of this study was to assess the interrelation between vascular ultrasonography (US) findings, histopathological data, and the expression of selected dysregulated microRNAs (miRNAs) in giant cell arteritis (GCA). The study included data on the clinical parameters, US measurements, and temporal artery biopsies (TABs) of 46 treatment-naïve patients diagnosed with GCA and 22 age-matched non-GCA patient controls. We performed a comprehensive comparative and correlation analysis along with generation of receiver operating characteristic (ROC) curves to ascertain the diagnostic performance of US examination parameters and selected miRNAs for GCA diagnosis. We showed significant differences in the US-measured intima-media thickness of the temporal arteries, the presence of a halo sign, and the presence of luminal stenosis between GCA-positive/TAB-positive, GCA-positive/TAB-negative, and non-GCA patients. Correlation analysis revealed significant associations between several histopathological parameters, US-measured intima-media thickness, and the halo sign. We found that the significant overexpression of miR-146b-5p, miR-155-5p, miR-511-5p, and miR-21-5p, and the under-expression of the miR-143/145 cluster, miR-30a-5p, and miR-125a-5p, coincides and is associated with the presence of a halo sign in patients with GCA. Notably, we determined a high diagnostic performance of miR-146b-5p, miR-21-3p, and miR-21-5p expression profiles in discriminating GCA patients from non-GCA controls, suggesting their potential utilization as putative biomarkers of GCA. Taken together, our study provides an insight into the US-based diagnostic evaluation of GCA by revealing the complex interrelation of clearly defined image findings with underlying vascular immunopathology and altered arterial tissue-specific miRNA profiles.

Association between Sperm Morphology and Altered Sperm microRNA Expression.

Evaluation of male infertility has been based on semen analysis for years. As this method can be subjective at times, there is a scientific tendency to discover stable and quantifiable biomarkers. This study included 28 couples who underwent an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. The couples were assigned into two groups, according to sperm morphology. Couples where the males were normozoospermic were placed in the control group (15 participants), while couples where males had teratozoospermia were placed in the study group (13 participants). Thirteen candidate miRNAs were selected for qPCR analysis, based on our literature search. We determined significant under-expression of nine miRNAs (miR-10a-5p/-15b-5p/-26a-5p/-34b-3p/-122-5p/-125b-5p/-191-5p/-296-5p and let-7a-5p) in spermatozoa from patients with teratozoospermia compared to the controls, whereas expression levels of four miRNAs (miR-92a-3p/-93-3p/-99b-5p/-328-3p) did not significantly differ between the study and control groups. The expression levels of all 13 included miRNAs were significantly positively correlated with each other and significantly positively associated with spermatozoa morphology, excluding miR-99b-5p. There were no other significant associations between miRNA expression and sperm quality parameters. Only expression levels of miR-99b-5p were significantly positively correlated with good-quality day 3 embryo rate (ρ = 0.546; p = 0.003), while other variables of the IVF/ICSI cycle outcome showed no significant associations with miRNA expression profiles. This is one of the rare studies providing an insight directly into miRNA profiles in regard to sperm morphology. We identified nine miRNAs that could serve as biomarkers of spermatozoa quality in regard to teratozoospermia.

Usefulness of high-risk human papillomavirus mRNA silver in situ hybridization diagnostic assay in oropharyngeal squamous cell carcinomas.

AIMS: The transcriptional activity of high-risk human papillomaviruses (HR-HPV) within oropharyngeal squamous cell carcinomas (OPSCC) has been linked to improved survival of patients. HR-HPV mRNA silver in situ hybridization (SISH) was evaluated on a cohort of OPSCC and compared with viral HPV DNA tests and p16 expression. Clinical outcomes of HPV-driven OPSCC and non-HPV related OPSCC were also studied. METHODS: We evaluated 67 OPSCC and 3 papillomas, obtained from 62 patients, for detection of HR-HPV DNA by PCR tests. The positive samples were additionally studied by the SISH method using three probes of HPV16, HPV18, and HP33, and for p16 expression detected by immunohistochemistry. SISH assays were evaluated for the presence/number and intensity of signals in cancer cells. Prognostic significance of HPV status in our cohort was evaluated with univariate and multivariate statistics. RESULTS: According to the HR-HPV PCR tests, 46 (69%) OPSCC cases were HPV positive, while three papillomas were negative. Of total 46 HPV-positive OPSCCs, 43 cases were also SISH-positive, while p16 overexpression was found in 45 of 46 HPV positive OPSCC cases. In OPSCC specimens, the sensitivity and specificity of the combined SISH probes (HPV16 and 33) were both 100.00%, when compared to HPV PCR. HPV positivity of the tumors appeared significant for predicting progression-free survival, cause specific survival and overall survival in a multivariate setting. CONCLUSIONS: The recently developed mRNA SISH methodology can detect HPV-driven OPSCCs without any additional test in 79% of cases. Positive SISH signals enable the visualization of viral transcripts required to recognize clinically relevant HPV infection. However, rare and tiny signals require an experienced pathologist to establish a consensus interpretation of results. The currently applied HR-HPV mRNA SISH analysis may serve as a groundwork for additional studies.

Inflammatory Cell Composition and Immune-Related microRNA Signature of Temporal Artery Biopsies From Patients With Giant Cell Arteritis.

Objectives: The 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). Methods: The 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. Results: Intense 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. Conclusion: Overall, 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.

Identification of microRNAs and their target gene networks implicated in arterial wall remodelling in giant cell arteritis.

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.

Association between histopathological changes and expression of selected microRNAs in skin of adult patients with IgA vasculitis.

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.

MicroRNA expression in the affected skin of adult patients with IgA vasculitis.

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.

Long Noncoding RNAs as Biomarkers in Cancer.

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: Biogenesis, Function, and a Role as Possible Cancer Biomarkers.

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.

Effect of Mycoplasma synoviae and lentogenic Newcastle disease virus coinfection on cytokine and chemokine gene expression in chicken embryos.

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-1ß, IL-6, IL-12p40, IL-16, IL-18, MIP-1ß (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-1ß, IL-2, IL-16, IL-21, XCL1, and MIP-1ß (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.