Belickienė, Violeta

This study examines five circulating microRNAs (miRNAs) (miR-29a, let-7i, miR-204, miR-142, and miR-328) potentially relevant to the diagnostic and/or prognostic significance of myopia. miRNA expression was analyzed using StepOnePlus™ RT-PCR. miRNA expression was evaluated in whole blood samples collected and stored using Tempus™ Blood RNA Tubes. The expression of five miRNAs was analyzed in 88 participants: 45 in the myopia group and 43 in the control (emmetropic) group. Heatmap analysis revealed differences in the expression patterns of miR-29a, let-7i, miR-204, and miR-142 when comparing individuals with myopia and controls. The intensity of miR-328 expression was similar between the myopia and control groups. A significant correlation was observed between miR-204 ΔCt and spherical equivalent. However, comparison of median values between different degrees of myopia and the control group did not reveal significant differences. The simultaneous evaluation of multiple circulating miRNAs may help to better understand biological processes associated with myopia.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor and non-motor symptoms, including cognitive decline and reduced quality of life. Identifying reliable biomarkers for disease progression and symptom severity remains a critical challenge. In this study, levels of oxidative stress-related microRNAs (miR-24-3p, miR-103a-3p, miR-320a-3p, miR-494-3p, miR-126-5p, and miR-543) within blood serum extracellular vesicles (EVs) were examined in a cohort of 93 PD patients to assess their associations with cognitive function, symptom severity, quality of life, and other clinical characteristics. The methods included microRNA extraction from blood serum EVs, followed by cDNA synthesis and RT-qPCR for expression analysis. Upregulation of miR-126-5p, as well as downregulation of miR-24-3p showed the strongest associations with symptom severity and cognitive decline, whereas downregulated miR-320a-3p levels correlated with patient-reported quality of life in PD patients. Downregulation of miR-103a-3p, and miR-543 expression showed slight associations with motor symptoms, cognitive function, and quality of life domains; however, some of these associations lacked statistical power. These findings indicate that specific microRNA expression profiles in extracellular vesicles are associated with PD symptom severity and progression, supporting their further evaluation as biomarkers in larger independent cohorts.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system characterized by considerable clinical heterogeneity. Its diagnosis and prognosis remain challenging – although clinical, radiological, and laboratory criteria are applied, reliable biomarkers capable of enabling earlier detection, predicting relapses, or assessing therapeutic efficacy are still lacking. Moreover, treatment effectiveness is often limited by inter-individual variability in therapeutic response and the complexity of disease progression. One promising class of biomarkers is microRNAs (miRNAs), small non-coding RNA molecules that regulate gene expression at the transcriptional or post-transcriptional level. However, due to their broad diversity, identifying biologically relevant miRNAs requires integration of in silico and experimental approaches. Our study consisted of two major stages: (1) in silico miRNA modeling and (2) an experimental sequencing phase. In the silico phase, we computationally derived predicted miRNA expression profiles in the context of MS. MiRNA sequencing was performed using the Illumina platform on extracellular vesicle samples isolated from blood (9 MS patients and 13 healthy controls). Differential expression analysis was conducted using the DESeq2 package to evaluate miRNA expression profiles between MS patients and healthy individuals. Overlapping miRNAs identified through both in silico prediction and sequencing were compared to assess their biological relevance to MS. Four miRNAs predicted in silico were confirmed in sequencing data. Among them, hsa-miR-19a-3p showed the highest expression change (log₂FoldChange > 1), while hsa-miR-17-5p, hsa-miR-139-5p, and hsa-miR-20a-5p exhibited moderate but significant alterations. KEGG analysis revealed enrichment in pathways of neurodegeneration – multiple diseases, amyotrophic lateral sclerosis (ALS), and the MAPK signaling pathway, suggesting shared molecular mechanisms between MS and other neurodegenerative disorders. The integration of in silico modeling with high-throughput sequencing analysis effectively identifies biologically relevant miRNAs in multiple sclerosis and highlights their potential as biomarkers involved in neurodegenerative and inflammatory signaling pathways.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons in the substantia nigra. PD usually leads to hallmark motor symptoms such as bradykinesia and resting tremor. Although current therapies can alleviate symptoms, the disease remains incurable. MiRNAs derived from extracellular vesicles (EV-derived) have shown great potential as biomarkers for various diseases, including PD. Therefore, finding miRNA biomarkers could enable early disease detection and support personalised treatment strategies. We aimed yo profile EV-derived miRNAs in Parkinson’s disease patients and healthy controls using next-generation sequencing (NGS) to identify potential biomarkers for the disease. EV-derived miRNAs were isolated from the blood serum of five PD patients and five healthy controls. The samples were then subjected to NGS. Sequencing data were analysed using principal component analysis (PCA), DESeq2 for differential expression and KEGG pathway analysis. PCA revealed two distinct clusters corresponding to healthy controls and PD patients. Differential expression analysis using DESeq2 identified ten miRNAs significantly deregulated in PD patients compared to controls. KEGG pathway analysis showed that these miRNAs are involved in pathways, like PD, Alzheimer’s disease, dopaminergic synapse, MAPK signaling, suggesting potential links to molecular mechanisms underlying PD. We identified ten miRNAs as potential biomarkers for Parkinson’s disease. These miRNAs were found to be involved in neurodegeneration-related pathways, suggesting their possible role in PD pathogenesis. Further studies with larger sample sizes are required to validate these findings and better characterize the expression patterns of these miRNAs.

Multiple sclerosis (MS) is a complex autoimmune disorder driven by diverse molecular, genetic, and immunological processes, and characterized by diverse clinical course forms. The identification of reliable biomarkers is essential for improving our understanding of MS disease mechanisms and for developing more advanced diagnostic and prognostic strategies. microRNA expression profiling represents one of the most promising approaches for revealing molecular alterations associated with MS pathogenesis. The aim of this study was to identify potential MS biomarkers by applying sequencingbased expression analysis and machine-learning methods using untreated samples. MiRNA sequencing was performed on samples from 9 MS patients and 13 healthy controls. Following data quality control, principal component analysis (PCA) was applied to evaluate sample structure and group separation. Differential expression analysis was conducted using DESeq2, with results visualized through a volcano plot and heatmap. In parallel, sPLS-DA was employed to identify features contributing most strongly to group discrimination. Molecules overlapping between DESeq2 and sPLS-DA results were further evaluated using boxplots and univariate logistic regression models. PCA analysis showed a clear separation between the MS and control groups based on miRNA expression. DESeq2 identified miRNAs that were significantly differentially expressed. sPLS-DA further narrowed down the set of features with strong ability to distinguish the groups. All miRNAs selected by sPLS-DA overlapped with those found by DESeq2, supporting their stability and biological relevance. Logistic regression showed that even single miRNAs from this set could significantly separate the two groups. The integrated DESeq2 and sPLS-DA analysis identified a group of miRNAs that were statistically different between MS patients and healthy controls and showed strong discriminatory ability. The overlapping miRNAs represent strong candidates for potential diagnostic or prognostic MS biomarkers. The analysis was performed using untreated samples, making the results more reliable and providing a solid basis for future validation studies.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder primarily affecting movement due to the progressive degeneration of dopaminergic neurons. It is characterized by motor symptoms such as tremors, bradykinesia and rigidity, which worsen over time. Extracellular vesicles (EVs) facilitate intercellular communication by transporting biomolecules, including microRNAs (miRNAs), which may reflect disease state and progression. Changes in EV miRNA expression could serve as valuable biomarkers for assessing PD severity and guiding treatment approaches. This study investigated the expression levels of specific EV miRNAs in the blood serum of PD patients at different stages of disease severity. EV miRNAs were extracted from blood serum, converted into cDNA, and analyzed using RT-PCR. The study included 88 PD patients with varying symptom severity. miRNA expression was assessed in relation to motor impairments such as bradykinesia, tremor, rigidity, dyskinesia, gait disturbances, and the "on-off" phenomenon, as well as disease onset and duration. Statistical analysis was conducted using GraphPad Prism 8. Among the analyzed EV miRNAs, miR-101-3p and miR-30b-5p showed a significant decline in expression as motor symptoms worsened. Patients experiencing more severe bradykinesia, tremor, balance, "on-off" phenomenon and gait difficulties exhibited the most pronounced downregulation of these miRNAs. Statistical analysis revealed a strong negative correlation between miRNA expression and general motor symptom severity, with miR-101-3p showing r = - 0.362 (p < 0.01) and miR-30b-5p exhibiting r = -0.411 (p < 0.001). However, no significant association was observed between miRNA expression and disease onset or duration. This study suggests that EV miRNAs could serve as biomarkers for Parkinson’s disease severity. The downregulation of miR-101-3p and miR-30b-5p correlates with worsening symptoms, highlighting their potential in disease monitoring and targeted treatment strategies.

Selenium is an essential microelement involved in antioxidant defence, redox regulation, and cellular signalling. Both its inorganic form, sodium selenite (Na₂SeO₃), and organic form, selenomethionine (SeMet), influence gene expression related to oxidative stress, apoptosis, and cell survival. The brain and liver, being highly vulnerable to oxidative stress, are critical tissues in understanding selenium’s biological effects. This study evaluates the impact of sodium selenite and selenomethionine on gene expression in these tissues, focusing on genes involved in redox regulation (MsrB1), apoptosis (Caspase-3), and cell cycle control (GADD45). The study was performed on 4-6-week-old BALB/c mice, which were divided into 4 experimental groups, that were given solutions of different concentrations of SeMet or Na₂SeO₃ (0.2 and 0.4 mg Se/kg body weight) for 8 weeks, and a control group that had free access to tap water. Then, mRNA was extracted from mouse liver and brain tissues and used to synthesize cDNA for RT-PCR analysis. The results show that MsrB1 expression in both brain and liver was higher in mice receiving 0.2 mg Se/kg of either SeMet or Na₂SeO₃ compared to the control group and those receiving 0.4 mg Se/kg. Caspase-3 expression in brain and liver was increased in mice receiving 0.2 mg Se/kg of Na₂SeO₃, but lower in those receiving 0.2 mg Se/kg of SeMet compared to the control group and those given 0.4 mg Se/kg of either selenium form. GADD45 expression in the brain and liver was elevated in mice given 0.2 mg Se/kg of Na₂SeO₃, while in SeMet-treated mice, GADD45 expression in the liver increased with higher concentrations, whereas in the brain, it decreased with increasing SeMet dosage. Overall, Na₂SeO₃ may promote stronger oxidative stress defence and apoptosis regulation, while SeMet could be more selective in its influence on apoptosis and cell cycle control, depending on tissue type.

Objectives* Demyelinating diseases are a group of pathologies characterized by damage to the myelin sheath and nerve cells. The myelin sheath plays a critical role in enhancing neural conduction velocity and conserving energy required for the propagation of action potentials. This category encompasses conditions such as multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, and multifocal neuropathy. Researchers are putting significant effort into identifying biomarkers that would help diagnose diseases more accurately and monitor their progression. Based on recommendations from the authors of previous publications to investigate a larger sample size for data reproducibility, we selected two miRNAs (miR-338-5p, miR-219a-5p, miR-146a-5p, miR-31-5p), which had significant associations with the studied pathologies. MiRNAs are short, non-coding, single-stranded molecules primarily involved in regulating gene expression through the suppression of transcription and translation. Materials and Methods In this study, circulating miR-338-5p and miR-219a-5p were investigated in the blood of 20 MS patients, and miR-146a-5p and miR-31-5p were examined in 12 CIDP and 9 MMN patients. Blood samples were collected into special Tempus blood tubes. RNA was isolated from blood using mirVana™ miRNA Isolation Kit (ThermoFisher Scientific (TFS), USA) and the cDNR synthesis was performed using the TaqMan® Advanced miRNA cDNA Synthesis Kit (TFS, USA). MiRNA expression was detected by using qRT-PCR method and the results were analyzed with ThermoFisher Cloud platform. Results The expression of miR-146a-5p and miR-31-5p in the peripheral venous blood of patients with chronic inflammatory demyelinating polyneuropathy and multifocal motor neuropathy compared to controls, while a significant difference was seen in serum extracellular vesicles. The analysis of differences in miR-338-5p expression showed that miR-338-5p expression was significantly higher in patients that taking 2nd-line treatment compared to those switched from 1st-line to 2nd-line treatment (p<0.05). Conclusions The results may assist in future studies aimed at evaluating the potential of these miRNA markers as diagnostic biomarkers for demyelinating diseases.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder globally, predominantly affecting people over 50 and characterized by symptoms like tremor and bradykinesia due to the degeneration of dopaminergic neurons. During PD progression, extracellular vesicles (EVs) carry biomolecules that reflect the disease state and transfer their cargo to recipient cells, potentially worsening the condition. MiRNAs within EVs may help distinguish disease stages, evaluate progression, and guide individualized treatment options. This study aimed to evaluate the expression levels of EV miRNAs in the blood serum of PD patients to differentiate between healthy individuals and PD patients with varying disease severity. EV miRNAs were isolated from blood serum, transcribed into cDNA, and quantified using RT-PCR. miRNA expression profiles were analyzed in 20 healthy controls (HC) and 88 Parkinson’s disease (PD) patients. We compared HC and PD groups and examined associations between miRNA expression and symptom severity, including bradykinesia, tremor, rigidity, dyskinesia, gait disturbances, nocturnal akinesia, the "on-off" phenomenon, orthostatic hypotension, as well as disease onset and duration. Long-term miRNA expression changes were also evaluated in patients undergoing gamma knife surgery (n=5) and deep brain stimulation (n=9) as treatments for PD. Statistical analyses were performed using GraphPad Prism 8. We identified significant differences in EV miR-126-5p and miR-543 expression among patient groups. ROC analysis demonstrated that the combination of miR-126-5p and miR-543 effectively differentiated PD patients from healthy controls, with an AUC of 0.982 (p<0.001). In PD patients, miR-126-5p was upregulated, whereas miR-543 was downregulated compared to healthy controls. Moreover, increased miR-126-5p expression correlated with greater severity of bradykinesia, orthostatic hypotension, and cognitive impairment (p<0.05). miR-543 expression was elevated in patients with more severe nocturnal akinesia but decreased in those with orthostatic hypotension (p<0.05). Disease onset and duration had no significant effect on miR-126-5p or miR-543 expression. Regarding surgical treatments, gamma knife surgery did not alter miRNA expression, while deep brain stimulation significantly increased miR-543 levels (p<0.05), suggesting a potential link between higher miR-543 expression and improved disease outcomes. In conclusion, the work demonstrates the importance of miRNA molecules in the context of PD. More detailed miRNA studies could be valuable in clinical practice for early PD diagnosis, enabling timely treatment and tracking disease progression.

Introduction Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide, primarily caused by the death of dopaminergic neurons [1]. The main symptoms of PD include bradykinesia, tremor, and rigidity [2]. Despite extensive research on the disease, there is still no cure to fully treat PD or reliable diagnostic tools capable of detecting it in its early stages [3]. Many researchers are now investigating the role of microRNAs (miRNAs) in biological processes, including their involvement in neurodegenerative diseases. miRNAs derived from extracellular vesicles (EVs) are being explored as potential biomarkers that could aid in early disease detection, assess disease progression and severity, and help tailor the most effective treatment based on an individual's miRNA profile [4]. MiR-543 regulates cell proliferation, migration, inflammation. In neurodegeneration, miR-543 induces α-synuclein aggregation, which leads to the death of dopaminergic neurons [5]. Aim The aim of this study was to measure the expression of EV miR-543 in the blood serum of PD patients and control group and to evaluate its potential correlation with clinical and demographic data. Methods Extracellular vesicle (EV) miR-543 was isolated from blood serum using the "exoRNeasy Midi Kit", transcribed into cDNA, and quantified via RT-PCR. Statistical analyses included Student's t-test (paired/unpaired), ANOVA, and Pearson’s correlation coefficient using GraphPad Prism 8, statistically significant if p<0.05. miR-543 expression was compared between 89 Parkinson's disease (PD) patients and a control group of 20 individuals without neurodegenerative disorders. The miR-543 profile was analyzed in relation to age, sex, disease onset, duration, symptom severity, and treatment methods. Results In this study participated 109 individuals: 20 older age individuals in whom neurodegenerative disorders were not detected, 37 received only medication, 39 underwent deep brain stimulation and 13 were treated with gamma knife surgery. The results indicated that miR-543 expression was downregulated in PD patients compared to the control group. There was no significant difference in miR-543 expression between different treatment methods. After gamma knife surgery miR-543 expression downregulated even more. MiR-543 expression after deep brain stimulation did not significantly change. When bradykinesia and tremor were more severe, miR-543 expression was upregulated; however, the data did not show a statistically significant difference. Sex, age, disease onset, duration didn’t show any significant impact to miR-543 expression. Conclusions In conclusion, miR-543 is significantly downregulated in Parkinson's disease and demonstrates potential as a diagnostic biomarker for the disease. However, further research is needed to assess the significance of miR-543 as a biomarker before considering its use in clinical trials.