Expression Levels Of microRNAs Research Articles

In situ imaging of intracellular miRNAs in tumour cells by branched hybridisation chain reaction.

The ability to visualise microRNA in situ is crucial for studying microRNAs, their microRNA-associated biological functions and disease diagnosis. Traditional fluorescence in situ hybridisation methods based on paraformaldehyde fixation of microRNAs suffer from release of microRNAs from cells, which limits the sensitivity of in situ hybridisation, making them unsuitable for the detection of small, low-abundance microRNAs. To reduce the loss, microRNAs were covalently cross-linked to proteins within cells by combining EDC and paraformaldehyde, and the target microRNA was used as the initiator chain for a branched hybridisation chain reaction to detect microRNA expression levels in situ. A simplified branched hybridisation chain reaction can be realised by coupling two hybridisation chain reaction circuits with a hairpin linker. Upon forming the primary hybridisation chain reaction product with extended sequence, this sequence reacts with the linker hairpin H3 to release the initiator sequence, resulting in the formation of numerous dendritic branched hybridisation chain reaction products. Imaging results show that this technique can detect microRNAs with high sensitivity and selectivity at both the single-cell and single-molecule levels. Compared with the traditional fluorescence in situ hybridisation technique, this method greatly improves the sensitivity and image resolution of in situ imaging detection. Therefore, we believe that the target-initiated branched hybridisation chain reaction based in situ detection method provides a reliable assay platform for analysing disease-related microRNA expression.

Are miR-26a and miR-26b microRNAs potent prognostic markers of gestational diabetes?

Gestational diabetes mellitus is a common public health problem, accompanied by complications for the mother and fetus. So, introducing new biomarkers to identify early diabetes is essential. As serum miRNAs are potentially appropriate markers, we investigated miR-26a and miR-26b expression levels in pregnant women with and without gestational diabetes. Demographic and clinical characteristics of 40 gestational diabetic patients and 40 healthy controls were assessed. The expression level of miR-26a and miR-26b microRNAs was measured by real-time PCR. Statistical analysis was done with GraphPad Prism software (version 8.4.3). The findings of this study showed that the expression level of miR-26a and miR-26b increased in women with gestational diabetes compared with healthy pregnant women, but the increase in expression was only significant for miR-26a (p < 0.05). According to the statistical and ROC curves, we suggest miR-26a as a potential biomarker for the early diagnosis of gestational diabetes mellitus.

Palmitic acid reduces the methylation of the FOXO1 promoter to suppress the development of diffuse large B-cell lymphoma via modulating the miR-429/DNMT3A axis

Diffuse large B-cell lymphoma (DLBCL) is characterized by significant treatment resistance. Palmitic acid (PA) has shown promising antitumor properties. This study aims to elucidate the molecular mechanisms by which PA influences DLBCL progression. We quantified the expression levels of microRNAs (miRNAs), Forkhead box protein O1 (FOXO1), and DNA methyltransferase 3A (DNMT3A) in both untreated and PA-treated DLBCL tumors and cell lines. Assessments were made of cell viability, apoptosis, and autophagy-related protein expression following PA administration. Interaction analyses among miR-429, DNMT3A, and FOXO1 were conducted using luciferase reporter assays and methylation-specific (MSP) Polymerase chain reaction (PCR). After transfecting the miR-429 inhibitor, negative control (NC) inhibitor, shRNA against DNMT3A (sh-DNMT3A), shRNA negative control (sh-NC), overexpression vector for DNMT3A (oe-DNMT3A), or overexpression negative control (oe-NC), we evaluated the effects of miR-429 and DNMT3A on cell viability, mortality, and autophagy-related protein expression in PA-treated DLBCL cell lines. The efficacy of PA was also tested in vivo using DLBCL tumor-bearing mouse models. MiR-429 and FOXO1 expression levels were downregulated, whereas DNMT3A was upregulated in DLBCL compared to the control group. PA treatment was associated with enhanced autophagy, mediated by the upregulation of miR-429 and downregulation of DNMT3A. The luciferase reporter assay and MSP confirmed that miR-429 directly inhibits DNMT3A, thereby reducing FOXO1 methylation. Subsequent experiments demonstrated that PA promotes autophagy and inhibits DLBCL progression by upregulating miR-429 and modulating the DNMT3A/FOXO1 axis. In vivo PA significantly reduced the growth of xenografted tumors through its regulatory impact on the miR-429/DNMT3A/FOXO1 axis. Palmitic acid may modulate autophagy and inhibit DLBCL progression by targeting the miR-429/DNMT3A/FOXO1 signaling pathway, suggesting a novel therapeutic target for DLBCL management.

Investigation of Antihypertensive Properties of Chios Mastic via Monitoring microRNA-21 Expression Levels in the Plasma of Well-Controlled Hypertensive Patients.

Hypertension is a chronic, multifactorial disease, leading to high cardiovascular morbidity and mortality globally. Despite the advantages of pharmaceutical treatments, natural products have gained scientific interest due to their emerging phytotherapeutic properties. Chios mastic is a natural Greek product, consisting of bioactive compounds which modify microRNAs' (small, expression-regulating molecules) expression. In this study, we investigated the antihypertensive properties of Chios mastic through the assessment of miR-21 levels. Herein, plasma samples of 57 individuals with hypertension, recruited for the purposes of the HYPER-MASTIC study, were analyzed. This was a clinical trial with Chios mastic supplements in which the patients were divided into groups receiving high and low mastic doses and placebo supplements, respectively. miR-21 was significantly upregulated in patients compared to normotensive individuals. Mean changes in miR-21 levels were statistically significant, after adjusting for sex and age, between the placebo and low-dose group and between the low- and high-dose group. Post-intervention miR-21 levels were positively associated with night-time systolic blood pressure, pulse pressure, and central systolic mean arterial pressure and negatively associated with night-time pulse wave velocity in the low-dose group. Our findings suggest a potential implication of miR-21 in the association of Chios mastic with night-time blood pressure measurements.

Using Extracellular miRNA Signatures to Identify Patients with LRRK2-Related Parkinson’s Disease

Background: Mutations in the Leucine Rich Repeat Kinase 2 gene are highly relevant in both sporadic and familial cases of Parkinson’s disease. Specific therapies are entering clinical trials but patient stratification remains challenging. Dysregulated microRNA expression levels have been proposed as biomarker candidates in sporadic Parkinson’s disease. Objective: In this proof-of concept study we evaluate the potential of extracellular miRNA signatures to identify LRRK2-driven molecular patterns in Parkinson’s disease. Methods: We measured expression levels of 91 miRNAs via RT-qPCR in ten individuals with sporadic Parkinson’s disease, ten LRRK2 mutation carriers and eleven healthy controls using both plasma and cerebrospinal fluid. We compared miRNA signatures using heatmaps and t-tests. Next, we applied group sorting algorithms and tested sensitivity and specificity of their group predictions. Results: miR-29c-3p was differentially expressed between LRRK2 mutation carriers and sporadic cases, with miR-425-5p trending towards significance. Individuals clustered in principal component analysis along mutation status. Group affiliation was predicted with high accuracy in the prediction models (sensitivity up to 89%, specificity up to 70%). miRs-128-3p, 29c-3p, 223-3p, and 424-5p were identified as promising discriminators among all analyses. Conclusions: LRRK2 mutation status impacts the extracellular miRNA signature measured in plasma and separates mutation carriers from sporadic Parkinson’s disease patients. Monitoring LRRK2 miRNA signatures could be an interesting approach to test drug efficacy of LRRK2-targeting therapies. In light of small sample size, the suggested approach needs to be validated in larger cohorts.

Comparison of microRNA expression levels in patients with schizophrenia before and after electroconvulsive therapy.

Exploring the role of microRNAs in the antipsychotic efficacy of electroconvulsive therapy (ECT) will contribute to understanding the underlying mechanism through which ECT exerts its therapeutic effects. The primary objective of this study was to identify microRNA alterations before and after ECT in patients with schizophrenia. We compared microarray-based microRNA profiles in peripheral blood from eight patients with schizophrenia before and after ECT and eight healthy controls. Then, we aimed to validate selected differentially expressed microRNAs in 30 patients with schizophrenia following a course of ECT, alongside 30 healthy controls by using quantitative real-time PCR (qRT-PCR). Microarray-based expression profiling revealed alterations in 681 microRNAs when comparing pre- and post-ECT samples. Subsequent qRT-PCR analysis of the selected microRNAs (miR-20a-5p and miR-598) did not reveal any statistical differences between pre- and post-ECT samples nor between pre-ECT samples and those of healthy controls. As neuroepigenetic studies on ECT are still in their infancy, the results reported in this study are best interpreted as exploratory outcomes. Additional studies are required to explore the potential epigenetic mechanisms underlying the therapeutic efficacy of ECT.

Protosappanin B enhances the chemosensitivity of 5-fluorouracil in colon adenocarcinoma by regulating the LINC00612/microRNA-590-3p/Golgi phosphoprotein 3 axis

Background5-fluorouracil (5-FU) is conventionally used in chemotherapy for colon adenocarcinomas. Acquired resistance of 5-FU remains a clinical challenge in colon cancer, and efforts to develop targeted agents to reduce resistance have not yielded success. Protosappanin B (PSB), the main component of Lignum Sappan extract, is known to exhibit anti-tumor effects. However, whether and how PSB could improve 5-FU resistance in colon cancer have not yet been established. In this study, we aimed to explore the effects and underlying mechanisms of PSB in 5-FU-induced chemoresistance in colon adenocarcinoma.MethodsForty-seven paired colon cancer tissue samples from patients who received 5-FU chemotherapy were collected as clinical samples. Two 5-FU resistant colon cancer cell lines were established for in vitro experiments. Reverse transcription-quantitative PCR (RT-qPCR) was performed to determine the mRNA and microRNA (miRNA) expression levels in colon adenocarcinoma tissues and cell lines. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were performed to evaluate cell proliferation and apoptosis, respectively.ResultsLINC00612 was highly expressed in colon adenocarcinoma samples and 5-FU resistant colon cancer cells. LINC00612 knockdown enhances 5-FU chemosensitivity in 5-FU resistant cells. Notably, PSB treatment attenuated LINC00612 expression in 5-FU resistant colon adenocarcinoma cells. Moreover, PSB treatment reversed the increase in LINC00612-induced 5-FU resistance. Mechanistically, LINC00612 specifically bound to miR-590-3p, which promoted 5-FU resistance in colon adenocarcinoma cells and attenuated the inhibitory effect of LINC00612 on GOLPH3 expression.ConclusionPSB attenuates 5-FU chemoresistance in colon adenocarcinoma by regulating the LINC00612/miRNA-590-3p/GOLPH3 axis.Graphical

#1749 Influence of high saline diet on myocardial remodeling, microRNA-21i and NFkB expression in myocardium

Abstract Background and Aims There is now increased interest in the study of morpho-functional myocardial changes in excess NaCl consumption, which develop independently of AD dynamics. Myocardial remodeling may involve microRNAs that modulate gene expression at the post-transcription level. However, their role in this process has not been fully explored. The purpose of the study is to assess the levels of expression of the nucleic transcription factor kV (NFkB), microRNA-21 and structural changes in the myocardium at long-term consumption of Wistar diet with high (8%) table salt content. Method Male Wistar rats (body mass 280.5 42.7 g) were divided into two groups of 10 animals. For 4 months, the control rats (NaCl) received a standard diet (0.34% NaCl), and the animals of the other group received a high salt diet (8% NaCl) (HS). Laboratory food varied only in NaCl content. 4 months later in rats systolic AD was measured, myocardial mass index (IMM) was evaluated, myocardial histology was performed (paraffin coloration - hematoxylin-eosin, Masson), relative expression levels of my RNA-determined 21 and NFkB in the myocardium, sodium content in the urine and blood serum. Statistical analysis used the t-Student criterion and the Mann-Whitney test. The differences were considered significant at p &amp;lt; 0.05. Results Excess NaCl (8%) in the diet had no effect on animal body weight (370.5 32.0 g in HS group, 393.0 21.4 g in NS group, p &amp;gt; 0.05) and systolic AD. In the HS-Group, the average AD was 134.5 8.9 mm old, and in the NS-Group - 134.8 5.2 mm old. (p &amp;gt; 0.05). However, HS-group IMM rats were 25% higher than NS-group rats (p &amp;lt; 0.05). The daily water consumption in the HS group was on average 33% higher than in the control, the daily diuresis also increased (10.5 5.7 ml against 5.7 2.9 ml in the NS-group, p &amp;lt; 0.001) and the sodium content in the urine (81.25.72 mmol/l against 161.18.57 mmol in the NS-01). There was no difference in the sodium content of the blood serum. Hypertrophy of cardiomyocytes, protein dystrophy, and increased wall thickness of arterial vessels (NS-20,42 3,53, HS-27,61 4,93, µm were identified in the HS-group; p &amp;lt; 0.01). The area of perivascular fibrosis in rats of the HS group (14876 ± 3803.7 µm2) was almost 1.8 times higher than that in the NS group (8426.0 ± 3012.2 µm2, p &amp;lt; 0.03). The thickness of cardiomyocytes (NS-12.97 1.53, HS-20.28 1.90 µm, p &amp;lt; 0.001) also increased in rats on the HS diet. In animals of the HS group, the relative expression levels of NFkB (more than 2 times) and microRNA-21 (almost 6 times) in the myocardium increased compared to the NS group. It can be assumed that the adverse effect on the cardiovascular system of high-salt diets is partially realized through NFκB-associated signaling pathways and the activation of microRNA-21. Conclusion Long-term use of a high-salt diet in Wistar rats results in myocardial remodeling unrelated to changes in AD levels. In this case, the high salt intake effects on the myocardium may be mediated by an increase in NFkBy and microRNA-21 expression levels.

#2229 miR-186, a potential noninvasive diagnostic biomarker of Membranous nephropathy in urinary microparticles

Abstract Background and Aims Membranous nephropathy (MN) is one of the most common causes of nephrotic syndrome, and is associated with podocyte damage and proteinuria. Invasive biopsy is the gold-standard diagnostic method for this disease. The use of specific biomarkers, such as microparticles (MPs) originating from damaged podocytes released in a patient's urine, has been proposed as an early, noninvasive, and repetitive diagnostic method. According to the previous studies, MPs derived from podocytes are present in the urine of patients with different glomerulonephritis, such as diabetic nephropathy, lupus nephritis, renal injury in preeclampsia, and renovascular hypertensive disorders. The analysis of urinary microparticle-derived miRNAs from patients with kidney diseases can help detect diagnostic biomarkers. This study aimed to analyze podocyte damage-related miRNAs in MN, miR-30c, miR-135, and miR-18 in the urinary MP of MN patients to identify appropriate candidate biomarkers for MN Method Urine samples from 30 patients with membranous nephropathy and 16 healthy individuals were collected to extract podocyte MPs using ultracentrifugation. Characterization of these particles was determined using the specific protein marker of MPs, TSG-101, and the podocyte-specific marker Nephrin, by western blotting, and SEM was used to check the size and morphology. The expression levels of microRNAs extracted from podocyte MPs, miR-30c, miR-135, and miR-186, were also evaluated by real-time PCR. Results The evaluation of the characteristics of MPs showed that there were many particles with a size of more than 100 nm in the urine sample, and there were higher levels of the specific markers TSG-101 and Nephrin in the urine samples of MGN patients than in the control samples (P &amp;lt; 0.001). The expression level of miR-186 was compared between the three studied groups and based on the results of the expression of this miRNA in the group of membranous nephropathy patients compared to healthy people group, there was a significant increase in expression (P = 0.000) Conclusion This study showed that although the expression levels of miR-186 in podocyte MPs samples extracted from the urine of patients with MN were significantly changed compared to the studied controls, more studies should be performed to determine the diagnostic values.

Investigating the Impact of Circulating miRNAs on Cardiovascular System Remodeling During Pregnancy

During pregnancy, women undergo significant remodeling of their cardiovascular system, a complex process involving various physiological adaptations to ensure an adequate blood supply to the developing fetus. An intriguing area of research focuses on the role of circulating microRNAs, particularly the primate-specific chromosome 19 miRNA cluster (C19MC) and the rodent-specific chromosome 2 miRNA cluster (C2MC), both of which exhibit high expression levels in the placenta. Extracellular vesicles, such as exosomes, play a crucial role in intercellular communication and may carry microRNAs that contribute to the physiological changes observed during pregnancy. However, the influence of circulating C19MC and C2MC microRNAs on maternal cardiovascular system remodeling remains unexplored. We hypothesized that the increased levels of circulating C2MC or C19MC microRNAs during pregnancy might play a role in the maternal cardiovascular system adaptations. To investigate this hypothesis, we conducted a study to determine whether circulating exosomes during pregnancy lead to elevated expression levels of C19MC or C2MC microRNAs in various organs, including the heart, kidney, liver, spleen, lungs, and brain. In our investigation, we utilized three distinct mouse models: wild-type (WT) mice expressing only C2MC, C2MC knock-out (KO) mice, and a novel humanized mouse model, which combined C2MC KO with either a single or double copy of the human C19MC. We extracted total RNA from the organs of these mice and performed cDNA synthesis. Subsequently, we measured the expression levels of two representative microRNAs, miR-517a and miR-466a, corresponding to C19MC and C2MC, respectively, in the organs of pregnant and non-pregnant mice using TaqMan RT-qPCR. Our results indicated no significant differences in the expression levels of miR-517a and miR-466a when normalized to the housekeeping gene snoRNA-202 across different organs in pregnant and non-pregnant mice. To comprehensively assess the expression patterns of all microRNAs within the C19MC and C2MC clusters in various organs of pregnant and non-pregnant mice, further investigation with a larger sample size and small RNA-Seq analysis is warranted. This approach will enable us to explore a broader spectrum of microRNAs and potentially identify specific candidates that may play a crucial role in the essential physiological process of maternal cardiovascular system remodeling. This study is supported by USF Cardiovascular Summer Undergraduate Research Fellowship (SURF) sponsored by the American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Circulating microRNAs and therapy-associated cardiac events in HER2-positive breast cancer patients: an exploratory analysis from NeoALTTO

PurposeThe relevance of cardiotoxicity in the context of HER2-positive breast cancer is likely to increase with increasing patient treatment exposure, number of treatment lines, and prolonged survival. Circulating biomarkers to early identify patients at risk of cardiotoxicity could allow personalized treatment and follow-up measures. The aim of this study is to examine the relationship between circulating microRNAs and adverse cardiac events in HER2-positive breast cancer patients.MethodsWe based our work on plasma samples from NeoALTTO trial obtained at baseline, after 2 weeks of anti-HER2 therapy, and immediately before surgery. Eleven patients experienced either a symptomatic or asymptomatic cardiac event. Circulating microRNAs were profiled in all patients presenting a cardiac event (case) and in an equal number of matched patients free of reported cardiac events (controls) using microRNA-Ready-to-Use PCR (Human panel I + II). Sensitivity analyses were performed by increasing the number of controls to 1:2 and 1:3. Normalized microRNA expression levels were compared between cases and controls using the non-parametric Kruskal–Wallis test.ResultsEight circulating microRNAs resulted differentially expressed after 2 weeks of anti-HER2 therapy between patients experiencing or not a cardiac event. Specifically, the expression of miR-125b-5p, miR-409-3p, miR-15a-5p, miR-423-5p, miR-148a-3p, miR-99a-5p, and miR-320b increased in plasma of cases as compared to controls, while the expression of miR-642a-5p decreases. Functional enrichment analysis revealed that all these microRNAs were involved in cardiomyocyte adrenergic signaling pathway.ConclusionThis study provides proof of concept that circulating microRNAs tested soon after treatment start could serve as biomarkers of cardiotoxicity in a very early stage in breast cancer patients receiving anti-HER2 therapy.

Promising two-dimensional rhenium diselenide nanosheets: Facile Preparation, characterization, in vivo tumor photoacoustic imaging and photothermal therapy

Nanoagent-mediated photothermal therapy (PTT) has been considered as an useful strategy for curing cancer. Nonetheless, identifying high-quality photothermal nanoagents with excellent biocompatibility testing, superior photothermal conversion efficiency (PTCE), and high treatment efficacy, is still a key dilemma for performing practical application in biomedicine field. In our work, a novel photothermal nanoagent named two-dimensional (2D) colloidal rhenium diselenide (ReSe2) nanosheets was exfoliated via a facile liqiud-assisted exfoliation approach. Owing to surface functionalization with poly-ethylene glycol (PEG), the obtained PEG-ReSe2 nanosheets demonstrated good solubility and excellent biocompatibility. Notably, the PEG-ReSe2 nanosheets showed excellent near infrared (NIR) photothermal activity. For the radiation of an NIR laser at 808 nm, an ultrahigh PTCE of 81.3% was demonstrated. Owing to its high Re element and good photoacoustic effect, the PEG-ReSe2 nanosheets can serve as good contrast nanoagents for in vivo photoacoustic (PA) imaging. Furthermore, the PEG-ReSe2 nanosheets acted as excellent photothermal agents (PTAs) for performing PTT. Thanks to high photothermal effects of PEG-ReSe2 nanosheets, in vitro photothermal effects showed that, the mortality rate of 4T1 cells was almost 100%. Meanwhile, in vivo experiments showed that mouse tumors were 100% ablated. More importantly, the PEG-ReSe2 nanosheets exhibited negligible levels of toxicity, as demonstrated by in vivo cytotoxicity assays and histological analysis. Finally, changes in micro-RNA expression level was proposed to assess PTT treatment efficacy of PEG-ReSe2 nanoagent. Interestingly, the miRNAs such as miR-125b-5p, miR-20a-5p, miR-214–3p, miR-221–3p, miR-212–3p and miR-27b-3p, were down-regulated. In addiiton, the miRNAs including miR-451a, miR200c-3p, and miR-let-7d-3p, were up-regulated. More importantly, these obtained miRNAs can work as typical indicators for screening PTT outcomes of PEG-ReSe2 nanosheets-involved PTT. Our research suggested that, the PEG-ReSe2 nanoagent may represent potential therapeutic nanoagents for in vivo photoacoustic imaging and cancer therapy.

An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

Near-infrared light-induced homogeneous photoelectrochemical biosensor based on 3D walking nanomotor-assisted CRISPR/Cas12a for ultrasensitive microRNA-155 detection

The dysregulation of microRNA (miRNA) expression levels is intricately linked to a myriad of human diseases, and the precise and delicate detection thereof holds paramount significance in the realm of clinical diagnosis and therapy. Herein, a near-infrared (NIR) light-mediated homogeneous photoelectrochemical (PEC) biosensor was constructed for miRNA-155 detection based on NaYF4: Yb, Tm@ZnIn2S4 (NYF@ ZIS) coupled with a three-dimensional (3D) walking nanomotor-assisted CRISPR/Cas12a strategy. The upconverted light emitted by the NYF in the visible and UV region upon NIR light excitation could be utilized to excite ZIS to produce a photocurrent response. The presence of target miRNA-155 initiated an amplification reaction within the 3D walking nanomotor, resulting in the production of multiple nucleic acid fragments. These fragments could activate the collateral cleavage capability of CRISPR/Cas12a, leading to the indiscriminate cleavage of single-stranded DNA (ssDNA) on ALP-ssDNA-modified magnetic beads and the subsequent liberation of alkaline phosphatase (ALP). The released ALP facilitated the catalysis of ascorbic acid 2-phosphate to generate ascorbic acid as the electron donor to capture the photogenerated holes on the NYF@ZIS surface, resulting in a positively correlated alteration in the photocurrent response. Under optimal conditions, the NIR light-initiated homogeneous PEC biosensor had the merits of good linear range (0.1 fM to 100 pM), an acceptable limit of detection (65.77 aM) for miRNA-155 detection. Considering the pronounced sensitivity, light stability, and low photodamage, this strategy presents a promising platform for detecting various other miRNA biomarkers in molecular diagnostic practice.

MiR-378a-3p and miR-491-5p as markers of xenon abuse in doping control

Xenon stimulates the synthesis of the hormone erythropoietin, which leads to improved oxygen supply to tissues, increased endurance and can be used by athletes to gain an undue advantage in competitions. The World Anti-Doping Agency (WADA) banned its use. The determination of xenon in biological fluids, in particular, in blood plasma samples, is difficult due to the narrow detection window. Its indirect detection is possible by changing in some blood parameters during a clinical analysis (RET%, HGB, HCT, etc.), however, this analysis is nonspecific and the use of other erythropoiesis-stimulating agents can lead to similar changes.Aims: The aim of the study was to search for long-term microRNA markers, the expression of which is specific and markedly altered by inhaled xenon.Methods: Quantitative real-time PCR was performed on CFX96 Bio-Rad analyser using miRCURY® LNA® miRNA SYBR® Green PCR Kit and panels for studying the expression profiles of mature microRNAs of the hypoxia signaling pathway miRCURY LNA™ miRNA Focus Panel.Results: Based on statistical data analysis, it was found that the expression of hsa-miR-378a-3p and hsa-miR-491-5p in blood plasma increases significantly (more than 70 times) when xenon inhalations are used as an erythropoiesis stimulator. Measurement of hematological parameters before and after inhalation showed no significant changes that could affect endurance or give competitive advantages.Conclusion: The evaluated difference in microRNA expression levels before and after administration of the xenon mixture (Xe/O2) makes hsa-miR-378a-3p and hsa-miR-491-5p potential candidates for the role of long-term markers of xenon abuse.

A highly sensitive Lock-Cas12a biosensor for detection and imaging of miRNA-21 in breast cancer cells

The expression levels of microRNA (miRNA) vary significantly in correlation with the occurrence and progression of cancer, making them valuable biomarkers for cancer diagnosis. However, their quantitative detection faces challenges due to the high sequence homology, low abundance and small size. In this work, we established a strand displacement amplification (SDA) approach based on miRNA-triggered structural “Lock” nucleic acid (“Lock” DNA), coupled with the CRISPR/Cas12a system, for detecting miRNA-21 in breast cancer cells. The “Lock” DNA freed the CRISPR-derived RNA (crRNA) from the dependence on the target sequence and greatly facilitated the extended detection of different miRNAs. Moreover, the CRISPR/Cas12a system provided excellent amplification ability and specificity. The designed biosensor achieved high sensitivity detection of miRNA-21 with a limit of detection (LOD) of 28.8 aM. In particular, the biosensor could distinguish breast cancer cells from other cancer cells through intracellular imaging. With its straightforward sequence design and ease of use, the Lock-Cas12a biosensor offers significant advantages for cell imaging and early clinical diagnosis.

Amplification-Free Analysis of Bladder Cancer MicroRNAs on Wrinkled Silica Nanoparticles with DNA-Functionalized Quantum Dots.

Bladder cancer (BC) occurrence and progression are accompanied by alterations in microRNAs (miRNAs) expression levels. Simultaneous detection of multiple miRNAs contributes to the accuracy and reliability of the BC diagnosis. In this work, wrinkled silica nanoparticles (WSNs) were applied as the microreactor for multiplex miRNAs analysis without enzymes or nucleic acid amplification. Conjugated on the surface of WSNs, the S9.6 antibody was adopted as the universal module for binding DNA/miRNA duplexes, regardless of their sequence. Furthermore, single-stranded DNA (ssDNA) was labeled with quantum dots (QDs) for identifying a given miRNA to form QDs-ssDNA/miRNA, which enabled the specific capture of the corresponding QDs on the wrinkled surface of WSNs. Based on the detection of fluorescence signals that were ultimately focused on WSNs, target miRNAs could be sensitively identified to a femtomolar level (5 fM) with a wide dynamic range of up to 6 orders of magnitude. The proposed strategy achieved high specificity to obviously distinguish single-base mutation sequences and possessed multiplex assay capability. Moreover, the assay exhibited excellent practicability in the multiplex detection of miRNAs in clinical serum specimens.

Evaluation of expression levels of microRNA processing elements in patients with sudden sensorineural hearing loss

BackgroundMicroRNAs have a significant role in the function and development of the hearing system. Idiopathic sudden sensorineural hearing loss (SSNHL) is a complicated disorder with no long-established reason. Since microRNAs play imperative roles in every aspect of the neural system, their dysregulation may contribute to the onset of SSNHL. The current study aimed to assess the expression patterns of microRNA processing elements (DROSHA, DICER, and DGCR-8) as the vital factors in microRNA biology that can affect the expression levels of microRNA. This study assessed DROSHA, DICER, and DGCR-8 mRNA expression levels in the peripheral blood mononuclear cells (PBMC) of 50 patients with SSNHL and 50 matched controls. After the isolation of PBMC, total RNA was extracted, and the expression levels of DROSHA, DICER, and DGCR-8 genes were evaluated using quantitative real-time PCR.ResultsThe results illustrated significant up-regulation of DICER and DGCR-8 genes in SSNHL patients at the mRNA level. Furthermore, despite no significant change in DROSHA level, DICER and DGCR-8 were significantly correlated with SSNHL. However, there was no significant correlation between these gene expressions and the clinicopathological features of patients.ConclusionThis study verified for the first time that the DGCR_8 and DICER mRNA expression levels were significantly up-regulated in patients with SSNHL, proposing that microRNAs and their processing pathways play key roles in the progression and development of SSNHL.

MicroRNAs as Potential Biomarkers of Mental Disorders: Diagnostic and Prognostic Implications

Background: microRNAs are small non-coding RNAs that play a crucial role in post-transcriptional regulation of gene expression. Recent studies indicate the involvement of microRNAs in the pathogenesis of various psychiatric disorders, which allows to consider them as diagnostic biomarkers and therapeutic targets.The aim of this literature review is to analyse curren research on the involvement of microRNAs in the formation of psychiatric disorders, focusing on their potential as diagnostic and prognostic biomarkers. Materials and methods: to find relevant publications, we searched eLibrary and PubMed databases using the following keywords and terms: microRNAs, mental disorders, psychological status, biomarkers, diagnosis, prognosis.Conclusion: numerous studies suggest that microRNAs are involved in the regulation of critical molecular pathways associated with psychiatric disorders, including neurotransmission, neurodevelopment, and synaptic plasticity. Dysregulation of certain microRNAs has been observed in various psychiatric disorders such as major depressive disorder, schizophrenia, bipolar disorder, anxiety disorders, autism spectrum disorders, attention-deficit/hyperactivity disorder and addictive disorders. It is highlighted that the investigated microRNAs have a high potential to be used as diagnostic biomarkers, opening up possibilities for early detection and personalised treatment strategies. The identification of specific microRNAs associated with different psychiatric disorders offers new opportunities to develop innovative diagnostic approaches and targeted therapeutic interventions. However, a number of challenges remain, including differences in microRNA expression levels and profiles due to the heterogeneity of psychiatric disorders, the need for standardisation of microRNA analysis protocols and their further validation in large-scale studies on different patient populations. Collaborative efforts between basic genetics researchers and clinicians are needed to harness the full potential of microRNA analysis for the diagnosis and treatment of psychiatric disorders.

Hsa-Mir-1 and Hsa-Mir-1229 expression levels in recurrent miscarriage patients before and after lymphocyte therapy in comparison to control groups

Background. Recurrent pregnancy loss is defined as at least two consecutive clinical pregnancy losses before 20th week of gestation. In this study, we investigated microRNA-1 and microRNA-1229 in recurrent miscarriage patients before and after lymphocyte therapy in comparison to control group. Methods. First, a written and informed consent will be obtained from the target population of this study, who are people with RPL referring to Waliasr International Hospital. According to the standard protocol, the number of 2×107 is injected subcutaneously to patients. Real-time PCR technique will be used to check the expression level of microRNA-1 and microRNA-1229. Results. The gene expression level of micro-RNA 1 in RPL women before of lymphocyte therapy was 2.030±1.445 and the gene expression level of the same micro-RNA after the lymphocyte therapy of the mother was 1.101±0.4780. Also, the expression level of micro-RNA 1229 in these women was reported to be 2.100±0.6296 before treatment and 1.247±0.9631 after treatment with lymphocytes. This level of gene expression in control group, for micro-RNA 1 and micro-RNA 1229 genes is 1.000±0.08334 and 1.000±0.08091, respectively. Conclusion. The results of our study showed a noticeable decrease in the gene expression of microRNAs under study, both microRNAs 1 and 1229, in women with a history of three consecutive injections of the father's subcutaneous lymphocytes. Practical Implications. This study can show the therapeutic effect of lymphocyte for patients with recurrent miscarriage so that this treatment can be used along with other treatment methods.