miRNA Levels Research Articles

A Dual-Cycle Isothermal Amplification Method for microRNA Detection: Combination of a Duplex-Specific Nuclease Enzyme-Driven DNA Walker with Improved Catalytic Hairpin Assembly

The association between microRNAs and various diseases, especially cancer, has been established in recent years, indicating that miRNAs can potentially serve as biomarkers for these diseases. Determining miRNA concentrations in biological samples is crucial for disease diagnosis. Nevertheless, the stem-loop reverse transcription quantitative PCR method, the gold standard for detecting miRNA, has great challenges in terms of high costs and enzyme limitations when applied to clinical biological samples. In this study, an isothermal signal amplification method based on a duplex-specific nuclease (DSN) enzyme-driven DNA walker and an improved catalytic hairpin assembly (CHA) was designed for miRNA detection. First, biotin–triethylene glycol-modified trigger-releasable DNA probes were conjugated to the streptavidin-coated magnetic beads for recognizing the target miRNA. The DSN enzyme specifically hydrolyzes DNA strands when the DNA probe hybridizes with the targeted miRNA. This recycling process converts the input miRNA into short trigger fragments (catalysts). Finally, three hairpins of improved CHA are driven by this catalyst, resulting in the three-armed CHA products and a fluorescence signal as the output. This dual-cycle biosensor shows a good linear relationship in the detection of miR-21 and miR-141 over the final concentration range of 250 fM to 50 nM, presenting an excellent limit of detection (2.95 amol). This system was used to detect miR-21 and miR-141 in MCF-7 and 22RV1 cells, as well as in 1% human serum. This system can be used to evaluate the expression levels of miRNAs in different biological matrices for the clinical diagnosis and prognosis of different cancers.

Plasma microRNA Levels After Electroconvulsive Therapy in Treatment-Resistant Depressed Patients.

Electroconvulsive therapy (ECT) is one of the most effective treatments for treatment-resistant depression (TRD), even though the molecular mechanisms underlying its efficacy remain largely unclear. This study aimed, for the first time, to analyze plasma levels of miRNAs, key regulators of gene expression, in TRD patients undergoing ECT to investigate potential changes during treatment and their associations with symptom improvement. The study involved 27 TRD patients who underwent ECT. Plasma samples were collected at baseline (T0) and 1 month after the last ECT session (T1), and miRNA analysis was conducted by qRT-PCR. We also performed gene prediction of miRNAs differentially expressed and KEGG pathway analysis. miR-95-3p, miR-194-5p, miR-324-3p, miR-195-5p, miR-19b-3p, miR-30c-5p, let-7i-5p, and miR-497-5p were nominally downregulated at T1. Changes in miR-324-3p and miR-30c-5p levels between T0 and T1 significantly correlated with symptom improvement. Among the predicted miRNA target genes of these 2 miRNAs, we noticed the presence of VEGF and SIRT1, whose expression regulation has been associated with the ECT mechanism of action in previous studies. The study's most relevant results are related to the correlation between reductions in miR-30c-5p and miR-324-3p and the improvement of symptoms in response to ECT, positioning these miRNAs as promising candidates for further studies. These findings support and extend previous clinical and preclinical research indicating a role of miRNAs in ECT mechanism of action. However, no significant effects in ECT miRNA modulation were observed, highlighting the need for future replications in broader samples to confirm these results.

Interaction between microRNA and KRAS in Glioblastoma.

Glioblastoma (GBM) characterized byits rapid progression and challenging prognosis, often featuring mutations in the Kirsten rat sarcoma virus (KRAS) gene, which is crucial for numerous cellular signaling mechanisms. Emerging research underscores a significant interaction between KRAS and microRNAs (miRNAs) in these cancers, with miRNAs playing key roles as both regulators and mediators within the KRAS signaling framework. The concept of oncogene-induced senescence (OIS) is explored as a protective mechanism against tumor development, examining how K-RAS signaling is meticulously adjusted to bypass senescence, thereby enhancing cell growth and survival. In this study, we identify certain miRNAs that directly impact KRAS through mRNA targeting or by influencing its downstream signaling cascades. In turn, pathways activated by KRAS can modify the levels of specific miRNAs, establishing a feedback loop that balances cell regulation and tumor progression. We propose a theoretical framework where these interactions are crucial for deciphering the molecular underpinnings of GBM, potentially paving the way for innovative treatment approaches that focus on the miRNA-KRAS connection.

LIN-67 functionally interacts with heterochronic miRNAs and regulates developmental timing in Caenorhabditis elegans.

Temporal regulation of gene expression is required for developmental transitions, including differentiation, proliferation, and morphogenesis. In the nematode Caenorhabditis elegans , heterochronic microRNAs (miRNAs) regulate the temporal expression of genes that promote animal development. The heterochronic miRNAs lin-4 and let-7 are required during different stages of larval development and are associated with the miRNA-specific Argonaute ALG-1. In this study, we have identified lin-67 as a heterochronic gene that negatively regulates lin-4 , let-7, and alg-1 . Loss of lin-67 function restores proper developmental timing and stage-specific gene expression to hypomorphic lin-4 and let-7 mutants. We found that loss of lin-67 resulted in a reduced number of seam cells, defects in alae formation, precocious expression of an adult-specific gene reporter, and sterility. LIN-67 contains a K homology (KH) RNA-binding domain and is a homolog of the Sam68-like splicing factor KHDRBS2. We show that LIN-67 localizes to the nucleus throughout animal development and is enriched in nuclear foci. Mutating the KH domain of LIN-67 abolished the nuclear localization of LIN-67, suggesting that the localization of LIN-67 is likely dependent on RNA-binding activity. We show that LIN-67 negatively regulates lin-4 miRNA levels and restores normal levels of let-7 to alg-1 mutants, which can, at least in part, explain how lin-67 suppresses alg-1 . Our data indicate that lin-67 is a novel heterochronic gene that regulates developmental timing and miRNA-dependent gene regulation in C. elegans .

Role of miRNA regulation in IGFBP-2 overexpression and neuronal ferroptosis: Insights into the Nrf2/SLC7A11/GPX4 pathway in Alzheimer's disease.

Alzheimer's disease (AD) is a common neurodegenerative disease with pathological features including amyloid plaque deposits and neurofibrillary tangles. In this study, the expressions of miRNA, IGFBP-2 and neuronal ferritin were detected by qPCR, Western blot and immunohistochemistry. The regulatory effects of miRNA on IGFBP-2 and neuronal ferritin were further verified by intervention experiments with miRNA mimics and inhibitors. Double luciferase reporter gene assay and RNA immunoprecipitation were used to investigate the interaction between miRNA and target genes. Finally, the effect of miRNA on Nrf2/SLC7A11/GPX4 pathway was evaluated by antioxidant enzyme activity and oxidative stress marker detection. The overexpression of IGFBP-2 was found to be significantly increased with the deposition of neuronal ferritin. Expression levels of specific mirnas were significantly down-regulated in AD models and negatively correlated with IGFBP-2 and neuronal ferritin expression. Intervention experiments with miRNA mimics and inhibitors have confirmed that these mirnas can regulate the expression of IGFBP-2 and neuronal ferritin. Further studies revealed that these mirnas affect antioxidant enzyme activity and oxidative stress levels by targeting key genes in the Nrf2/SLC7A11/GPX4 pathway, thereby regulating the deposition of neuronal ferritin.

The miRNA-mRNA Regulatory Network in Human Hepatocellular Carcinoma by Transcriptomic Analysis From GEO.

Bioinformatics analysis of hepatocellular carcinoma (HCC) expression profiles can aid in understanding its molecular mechanisms and identifying new targets for diagnosis and treatment. In this study, we analyzed expression profile datasets and miRNA expression profiles related to HCC from the GEO using R software to detect differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs). Common DEGs were identified, and a PPI network was constructed using the STRING database and Cytoscape software to identify hub genes. The reduced levels of tumor suppressor miRNAs or down regulated DEmiRs may be increased levels of oncogenes, the oncomirs or up regulated DEmiRs may be decreased levels of tumor suppressor genes in cancerous cells. According to this strategy, increased and decreased DEGs, also increased and decreased DEmiRs were selected. The multimir package was employed to predict target genes for DEmiRs then DEmiRs-hub gene network created. We identified approximately 1000 overlapping DEGs and 60 DEmiRs. Hub genes included RRM2, MELK, KIF11, KIF23, NCAPG, DLGAP5, BUB1B, AURKB, CCNB1, KIF20A, CCNA2, TTK, PBK, TOP2A, CDK1, MAD2L1, BIRC5, ASPM, CDCA8, and CENPF, all associated with significantly worse survival in HCC. miR-224, miR-24, miR-182, miRNA-1-3p, miR-30a, miR-27a, and miR-214 were identified as important DEmiRs with targeting more than six hub genes. Generally, our findings offer insight into the interaction of hub genes and miRNAs in the development of HCC by bioinformatics analysis, information that may prove useful in identifying biomarkers and therapeutic targets in HCC.

Dicer inhibition delays wound closure by increasing SUZ12 levels and regulating ITGAV levels in keratinocytes.

Delayed wound closure is a significant hallmark associated with diabetes. A previous study from our laboratory identified decreased levels of Dicer and miRNAs together with altered levels of wound healing genes in the wounded tissues of diabetic rats. Comprehensive regulators of these wound healing genes mapped onto the PRC2 (polycomb repressive complex 2) complex. Here we show that Dicer inhibition increases the transcript levels of core components of the PRC2 complex, namely Suz12 (suppressor of zeste 12) and Ezh2 (enhancer of zeste 2) and of Mtf2 (metal response element-binding transcription factor 2), its additional subunit, and elevates H3K27me3 levels in HaCaT cells. Such patterns of increase were also observed in the wounded tissues of diabetic rats as compared to those of normal rats. In a scratch assay in HaCaT cells, while Dicer inhibition significantly prevented wound closure, this was rescued by Suz12 siRNA but not by Ezh2 inhibition, suggesting that Suz12 mediates the effects of Dicer siRNA in these cells. In addition, as compared to scramble transfected cells, Dicer siRNA decreased the levels of integrin alphaV (Itgav), that is extensively implicated in the process of wound healing and this effect was rescued in the presence of Suz12 siRNA. Itgav harbors potential histone methylation marks across the gene length and Dicer inhibition, by increasing PRC2-mediated H3K27 methylation on Itgav, possibly decreases its transcription that subsequently impairs wound closure. These data put forth novel aspects of delayed wound closure as seen during diabetes and might be a potential target for therapeutic intervention.

Micro-RNA Signature in CSF Before and After Autologous Hematopoietic Stem Cell Transplantation for Multiple Sclerosis.

MicroRNAs (miRNAs) are regulators of gene expression and have been reported to be dysregulated in people with multiple sclerosis (pwMS). Autologous hematopoietic stem cell transplantation (aHSCT) is an immune-ablative treatment intervention for pwMS. Currently, it is unknown if aHSCT affects expression levels of miRNAs in CSF. We explored the ability of circulating miRNA to discriminate between pwMS and healthy controls (HCs) and investigated whether these miRNAs were affected by treatment with aHSCT. Using quantitative reverse transcription PCR, 87 miRNAs were analyzed in CSF samples of a discovery cohort (baseline: 4 & HC: 4). The top 22 miRNAs discriminating between pwMS and HCs were then analyzed in 187 CSF samples of a validation cohort (pwMS: 50, HC: 32). Samples, failing quality control or being follow-ups to baseline samples with quality control issues, were excluded from further analyses. The remaining 133 samples (HC: 29, MS: baseline: 33, 1 year: 30, 2 years: 26, 3-5 years: 15) were analyzed for expression of the top 22 miRNAs. Twelve miRNAs were dysregulated in pwMS compared with HC (q < 0.05). Associations with clinical and analytical parameters were observed in relation to all 12 miRNAs; however, a cluster of 4 miRNAs (miR-16-5p, miR-21-5p, miR-150-5p, and miR-146a-5p) with strong correlations (r > 0.60, p < 0.001) with multiple parameters was identified. Of the 12 miRNAs, 8 were differentially expressed in pwMS with gadolinium-enhancing lesions at baseline and 4 by prior disease-modifying treatment class (p < 0.05). These 4 miRNAs correlated strongly with each other, decreased after aHSCT, and remained low throughout the follow-up period (p < 0.05). Target and pathway analysis of these revealed association with biological processes affecting cytokine production, inflammatory response, and regulation of myelin maintenance. miRNAs are dysregulated in CSF from pwMS and particularly in patients with less effective treatments and/or higher inflammatory disease activity. A 4-miRNA signature with elevated expression of miR-16-5p, miR-21-5p, miR-150-5p, and miR-146a-5p was recurring in multiple analyses. After intervention with aHSCT, the expression levels approached the levels of the HCs, suggesting a potent treatment effect.

Validation of miR-21, miR-31, miR-34A, and miR-203 Changes by Quantitative Polymerase Chain Reaction in Isolated Keratinocytes.

MiRNAs play integral roles in diverse cellular functions, and their dysregulation is central to various pathological processes. Thyroid hormone (TH) is indispensable for numerous physiological processes, and it has been shown that multiple miRNAs regulate TH signaling in various tissues.This chapter describes a method for validating changes in miRNA observed through RNAseq. The method involves measuring the expression of mature miRNA levels in keratinocytes using the stem-loop real-time PCR. This method utilizes stem-loop RT primers to create cDNA for specific miRNAs in a single RT reaction. The keratinocytes used are derived from the total skin of four-day-old wild-type controls and double thyroid receptors knock-out mice (dTRKO).

LRET-Based Simultaneous Detection of Dual miRNAs via Multitrap Optical Tweezers Assisted Suspension Array Tagged by Two Different Luminescent Quenchable UCNPs Combining CRISPR/Cas12a Amplification.

Nowadays, optical tweezers play a vital role not only in optical manipulation but also in bioassay. As principal optical trapping objects, microbeads can combine optical tweezers with suspension array technology, with amply focused laser beams and adequately concentrated tags contributing to highly sensitive detection. In view of the inefficiency of conventional single-trap optical tweezers, multitrap systems are developed. Here, green- and blue-emitting core-shell-shell upconversion nanoparticles (UCNPs) are adopted to encode microbeads and determine dual miRNAs, with the internal shells leading the luminescence process to facilitate quenching through luminescence resonance energy transfer (LRET). Utilizing the trans cleavage of CRISPR/Cas12a, quenched luminescence signals are recovered and amplified, causing further enhanced detection sensitivity. Ultimately, limits of detection (LOD) of 17 and 22 aM are obtained with excellent specificities verified. Furthermore, dual miRNAs from MCF-7, A549, and MCF-10A cells are extracted and detected, with results consistent with those obtained by PCR. Notably, miR-155 in MCF-7 and A549 cells is detectable at the single-cell level. Thus, the differences in the measured miRNA levels between MCF-7 and MCF-10A cells imply the potential of this method to discriminate breast cancer cells from epithelial cells despite the difficulty in distinguishing different cancer cells due to similar miRNA levels.

Methodological Assessment of ExoGAG for Isolation of Cerebrospinal Fluid Extracellular Vesicles as a Source of Biomarkers.

Extracellular vesicles (EVs) in cerebrospinal fluid (CSF) represent a valuable source of biomarkers for central nervous system (CNS) diseases, offering new pathways for diagnosis and monitoring. However, existing methods for isolating EVs from CSF often prove to be labor-intensive and reliant on specialized equipment, hindering their clinical application. In this study, we present a novel, clinically compatible method for isolating EVs from CSF. We optimized the use of ExoGAG, a commercially available reagent that has been tested in plasma, urine and semen, and compared it directly with differential ultracentrifugation using Western blotting, protein quantification, nanoparticle tracking analysis, and cryogenic electron microscopy. Additionally, we analyzed the presence of specific microRNAs (miRNAs) known to be present in CSF-derived EVs. Our data demonstrate that ExoGAG is an effective method for isolating EVs from CSF, yielding a higher amount of EVs compared to traditional ultracentrifugation methods, and with comparable levels of specific miRNAs. In conclusion, the use of ExoGAG in a clinical setting may facilitate the testing of biomarkers essential for tracking brain pathology in CNS diseases.

Relationship between circulating miRNA-222-3p and miRNA-136-5p and the efficacy of docetaxel chemotherapy in metastatic castration-resistant prostate cancer patients

BackgroundMetastatic castration-resistant prostate cancer is the most dangerous stage of prostate cancer, with a high mortality rate. Docetaxel chemotherapy is one of the most effective treatment methods currently, but some patients do not respond to chemotherapy. To avoid unnecessary toxicity in non-responders, this study explores the potential of circulating microRNAs as early biomarkers of docetaxel response in patients with metastatic castration-resistant prostate cancer.MethodsPC3 cells and DU145 cells were divided into the control, NC mimics, and miRNA-136-5p-mimics groups. Cell viability was measured using the CCK-8 assay. Cell apoptosis was determined by flow cytometry. Cell migration and invasion abilities were evaluated using the Transwell assay. Real-time quantitative PCR was used to measure the miRNA levels in cells and peripheral blood of patients. The miRNA-136-5p target genes were predicted by using the PITA, TargetScan, and miRanda databases. The target genes were analyzed with KEGG pathway analysis.ResultsIn both PC3 and DU145 cells, the miRNA-136-5p-mimics group exhibited significantly increased cell survival rates, migration and invasion numbers, and significantly decreased apoptosis rates than the control group (p < 0.05). The miRNA-222-3p and miRNA-136-5p levels were significantly increased in docetaxel-resistant PC3 and DU145 cells (p < 0.05). The levels of circulating miRNA-222-3p and miRNA-136-5p were significantly associated with docetaxel treatment (p < 0.05). Higher levels of miRNA-222-3p were observed in non-responsive patients (p < 0.05). The area under the curve for miRNA-222-3p was 0.76 (95%CI: 0.55–0.97), indicating its effectiveness as a predictive factor for non-responsive patients to docetaxel. Patients with high expression of miRNA-34c-5p after docetaxel chemotherapy had shorter overall survival times (P < 0.05). Bioinformatics analysis identified 110 potential target genes of miRNA-136-5p. KEGG revealed that these genes were mainly distributed in three pathways. Among them, the PI3K-AKT pathway was closely related to the metastasis of prostate cancer cells.ConclusionOur study demonstrates that miRNA-136-5p promotes the proliferation and invasion of PC3 and DU145 cells while inhibiting apoptosis. Circulating miRNA-222-3p may serve as a biomarker for early therapeutic response to docetaxel, and further clinical investigation is warranted. Additionally, miRNA-136-5p may have anti-cancer effects during docetaxel chemotherapy in metastatic castration-resistant prostate cancer.

Prospective use of miRNAs as biomarkers in the diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia in the aging population. Pathogenic processes related to the accumulation of amyloid plaques (Aβ) and intracellular neurofibrillary tangles (NFTs) begin during the asymptomatic stage long before the onset of deterioration in cognitive functions and neurodegeneration, which makes rapid diagnosis and treatment difficult. Although biochemical diagnostic markers isolated from the body fluids of AD patients are currently used, scientists are engaged in research into molecular biomarkers that will significantly accelerate the diagnosis long before the first clinical symptoms appear. The research presented here focused on microRNAs (miRNAs), small, non-coding RNA molecules that are involved in the regulation of the post-transcriptional expression of many genes. A review of the literature revealed that miRNAs play an important role in regulating the expression of genes involved in the pathophysiological mechanisms of AD. Changes in the levels of miRNAs in a patient's body fluids can be used for rapid diagnosis. Original scientific articles published between 2014 and 2023 describing clinical and experimental studies on the role and expression levels of various miRNAs were selected from scientific databases such as PubMed, NCBI, Science Direct, and Google Scholar. The selected miRNAs were divided into 2 groups based on their expression level in AD: those with increased expression and those with decreased expression. A review of the latest scientific reports confirms that miRNAs may be a promising source of non-invasive and widely available biomarkers. Additionally, their modulation may prove to be an effective therapeutic strategy in AD.

Exploring microRNA signatures in pediatric non-infectious uveitis: meta-analysis and molecular profiling of patient samples.

To find a distinct non-coding RNA characteristic for idiopathic uveitis in the pediatric population. To explore the autoimmune-related miRNA expression profile in pediatric patients with idiopathic uveitis (IU) and juvenile idiopathic arthritis-associated uveitis (JIA-AU) and find a common molecular background for idiopathic uveitis and other autoimmune diseases. The expression levels of miRNAs were analyzed by quantitative real-time PCR using serum samples from patients with idiopathic uveitis (n = 8), juvenile idiopathic arthritis-associated uveitis (n = 7), and healthy controls. We selected the most promising miRNAs from the original research papers: miR-16-5p, miR-26a-5p, miR-145-5p, and miR-451a as markers for juvenile idiopathic arthritis; miR-23a-3p, miR-29a-3p, miR-140-5p, miR-193a-5p, and miR-491-5p for uveitis in the adult population; and miR-125a-5p, miR-146a-5p, miR-155-5p, miR-223-5p, and miR-223-3p characteristic for both diseases and confirm their expression changes in serum from children with idiopathic uveitis. We comprehensively reviewed the literature enrolling the papers that met the inclusion criteria (miRNA and non-infectious uveitis/juvenile idiopathic arthritis) and performed target prediction analysis of appoint miRNAs. It additionally confirmed that altered miRNAs target the immunologically involved genes. Immunological-involved miRNAs such as miR-146a-5p and miR-155-5p show diverse expression levels in different patients as they interact with multiple targets. miR-204-5p is downregulated in both patient groups compared to healthy controls. miR-204-5p and miR-155-5p are candidates for molecular markers of autoimmune uveitis. We did not identify the miRNAs specific only to idiopathic uveitis, but for the first time in the pediatric population, we confirmed that this disease entity shares a molecular basis with other autoimmune diseases. Further studies are required to elucidate the molecular interactions among miRNAs, cytokines, and transcription factors within the intricate immune response, particularly in the eye.

Evaluation of some microRNAs dysregulation in obesity and obesity-related hypertension

Background MicroRNAs (miRNAs) are implicated in regulating obesity, but clinical trials have yielded conflicting results. Objective This study aimed to investigate the relationships between circulating levels of miRNA-221 and miRNA-222 in obesity and hypertension-related obesity in humans. This may shed light on the pathogenic pathways controlling obesity and provide noninvasive molecular indicators to identify and predict the disease. The relationships between circulating levels of the above miRNAs and variables related to adiposity and lipid profiles were further investigated. Patients and methods Using a quantitative real-time P technique, the expression levels of circulating miRNAs were determined in serum samples from 65 obese patients (35 without and 30 with hypertension) and 45 age-matched and sex-matched normal-weight individuals. Results and conclusion MiRNAs (221 and 222) have been shown to be differentially expressed in the sera of obese patients compared to controls. In addition, obese hypertensive patients were shown to have higher serum levels of miRNA-222 and miRNA-221 than healthy controls. Serum miRNA-221 was correlated with BMI, cholesterol, triglycerides, and low-density lipoprotein cholesterol. Thus, circulating levels of miRNA-221 and miRNA-222 inease in obesity and obesity-associated hypertension. These miRNAs may serve as markers for obesity and obesity-induced hypertension.

Circulating miR-23b-3p, miR-30e-3p, and miR-205-5p as Novel Predictive Biomarkers for Ramucirumab-Paclitaxel Therapy Outcomes in Advanced Gastric Cancer.

Angiogenesis inhibition treatments are limited and are often too late for advanced gastric cancer (GC) patients, in whom its efficacy is reduced. New molecular biomarkers are needed to optimize therapy regimens. In regard to this framework, circulating miRNAs, with high sensitivity and specificity, could be useful biomarkers of GC. The present longitudinal study was focused on analyzing the expression levels of a blood miRNA signature in a cohort of 40 patients receiving second-line therapy combining Ramucirumab and Paclitaxel, stratified based on their Progression-Free Survival (PFS). Using differential and bioinformatic analysis, miR-205-5p, miR-30e-3p, and miR-23b-3p were selected as possible predictive biomarkers, with the results showing that they were more highly expressed in patients exhibiting longer PFS and that they were involved in modulating angiogenesis. Furthermore, patients with longer PFS showed a progressive and significant decrease in the selected miRNA to minimal levels. The loss of the protective effect and the increased expression of the hypothetical targets, including angiopoietin-2, were then observed. The hypothesis was supported by the inverse correlation found for miR-205-5p and angiopoietin-2. Circulating levels of miR-205-5p were protective (HR = 0.37, p = 0.02) and patients with higher baseline miRNA levels had longer OS (12.47 vs. 9.00 months). Our findings suggest that these three miRNAs may be novel candidates as non-invasive predictive markers of therapy outcomes.

MiR472 Deficiency Enhances Arabidopsis thaliana Defense Without Reducing Seed Production.

After having co-existed in plant genomes for at least 200 million years, the products of microRNA (miRNA) and nucleotide-binding leucine-rich repeat protein (NLR) genes formed a regulatory relationship in the common ancestor of modern gymnosperms and angiosperms. From then on, DNA polymorphisms occurring at miRNA target sequences within NLR transcripts must have been compensated by mutations in the corresponding mature miRNA sequence. The potential evolutionary advantage of such regulation remains largely unknown and might be related to two nonexclusive scenarios: (i) miRNA-dependent regulation of NLR levels might prevent defense mis-activation with negative effects on plant growth and reproduction or (ii) reduction of active miRNA levels in response to pathogen-derived molecules (pathogen-associated molecular patterns [PAMPs] and silencing suppressors) might rapidly release otherwise silent NLR transcripts for rapid translation and thereby enhance defense. Here, we used Arabidopsis thaliana plants deficient for miR472 function to study the impact of releasing its NLR targets on plant growth and reproduction and on defense against the fungal pathogen Plectosphaerella cucumerina. We show that miR472 regulation has a dual role, participating both in the tight regulation of plant defense and growth. MIM472 lines, with reduced active miR472, are more resistant to pathogens and, correlatively, have reduced relative growth compared with wild-type plants, although the end of their reproductive phase is delayed, exhibiting higher adult biomass and similar seed yield as the wild-type. Our study highlights how negative consequences of defense activation might be compensated by changes in phenology and that miR472 reduction is an integral part of plant defense responses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Circulating microRNAs as a Prognostic Tool to Determine Treatment Efficacy in Lung Cancer Patients Undergoing Pembrolizumab PD-1 Blockade Immunotherapy.

Background: Pembrolizumab has recently emerged as a PD-1 blockade immunotherapy treatment for lung cancer. It is critical that such treatment strategies for lung cancer should be chosen not only on the basis of histopathological features and the expression of targetable cell surface proteins (such as PD-1), but should rather be selected based on other determinants of treatment success or risk factors for poor prognosis. One method to forecast cancer trajectory is the identification of biomolecular signatures such as microRNAs (miRNAs), non-protein-coding RNA molecules that play a regulatory role in gene expression by modulating the translation or stability of messenger RNA. Methods: To find out which miRNAs have an important influence on anti-PD-1 treatment outcomes, we evaluated miRNA levels in sera from 38 lung cancer patients undergoing 3 months of pembrolizumab treatment. We selected a panel of miRNAs previously shown to be involved in lung cancer or PD-1 signaling and performed qPCR analysis. Results: Overall, we observed a significant decrease in the levels of miR126-5p (4-fold), let-7a (5-fold), miR133a-3p (4-fold), miR3615 (2-fold), miR4516 (3-fold), miR16 (3-fold), miR34c-5p (2-fold), miR20b-5p (5-fold), miR106b-5p (5-fold), miR146a-5p (3-fold) and miR181b-5p (3-fold) in response to treatment indicating effectiveness of immunotherapy. Within our selected panel of miRNAs, we identified two markers relevant to cancer prognosis: miR-217, which is negatively associated with patient survival, and let-7a, which is positively associated with patient survival. Conclusions: Our findings suggest that circulating miRNAs can be used for future treatment evaluation and lung cancer prognosis, with potential as therapeutic targets.

MicroRNA Profiling of PRELI-Modulated Exosomes and Effects on Hepatic Cancer Stem Cells.

The increasing incidence and mortality rates of liver cancer have heightened the demand for the development of effective anticancer drugs with minimal side effects. In this study, the roles of exosomes derived from liver cancer stem cells (LCSCs) with PRELI (Protein of Relevant Evolutionary and Lymphoid Interest) modulation and their miRNAs were investigated to explore their therapeutic properties for liver cancer. Various techniques, such as miRNA profiling, microRNA transfection, overexpression, flow cytometry, Western blotting, and immunocytochemistry, were used to evaluate the effects of exosomes under PRELI up- and downregulation. Downregulated PRELI cellular exosomes (DPEs) reduced the levels of five markers-CD133, CD90, CD24, CD13, and EpCAM-in LCSCs, with the exception of OV-6. Conversely, upregulated PRELI cellular exosomes (UPEs) significantly increased the expression of CD90, CD24, and CD133 in NHs, with the maximum increase in CD24. PRELI upregulation altered expression levels of miRNAs, including hsa-miR-378a-3p (involved in stem-like properties), hsa-miR-25-3p (contributing to cell proliferation), and hsa-miR-423-3p (driving invasiveness). Exosomes with downregulated PRELI inhibited the AKT/mTORC1 signaling pathway, whereas LCSCs transfected with the candidate miRNAs activated it. Additionally, under PRELI upregulation, exosomes showed increased surface marker expression, promoting cancer progression. The modulation of PRELI in LCSCs affected miRNA expression significantly, revealing candidate miRNA targets for liver cancer treatment. Exosomes with PRELI downregulation show potential as a novel therapeutic strategy. Consequently, this study proposes the potential of PRELI-induced exosomes and the three miRNAs as a liver anticancer therapeutic candidate.

Downregulation of MGLL and microRNAs (miR-302b-5p, miR-190a-3p, miR-450a-2-3p) in non-small cell lung cancer: potential roles in pathogenesis

Genes involved in lipid metabolism have been considered potential therapeutic targets in lung cancer because lipid metabolism is severely disrupted in this cancer. Monoglyceride lipase (MGLL) is a lipolytic enzyme that converts monoacylglycerides to fatty acids and glycerol. MicroRNAs (miRNA), one of the most important epigenetic regulators of gene expression, are also considered potential biomarkers in diagnosing, treating, and prognosis lung cancer. This study aimed to investigate the potential effects of MGLL and related miRNAs (miR-302b-5p, miR-190a-3p, miR-450a-2-3p) in the pathogenesis of non-small cell lung cancer (NSCLC) by examining their expression levels and regulatory mechanisms. We analysed the expression levels of MGLL and miRNAs in 30 NSCLC and 20 non-cancerous tissues by qPCR. We performed in silico analyses to determine the biological functions of MGLL and miRNAs in NSCLC. A protein-protein interaction (PPI) network was constructed for MGLL, and gene ontology (GO) analysis, and the interacting genes were analysed using the TCGAnalyzer tool. Our study showed that the expression levels of MGLL, miR-302b-5p, miR-190a-3p and miR-450a-2-3p were significantly decreased in NSCLC tissues (p < 0.05). Also, according to TCGAnalyzer, MSRB3, HTR4, and FCER1G genes were downregulated genes for NSCLC. We showed that miR-302b-5p, miR-190a-3p, and miR-450a-2-3p significantly regulate the TGF-β signalling pathway. In conclusion, this study provides evidence for the potential role of MGLL and microRNAs (miR-302b-5p, miR-190a-3p, miR-450a-2-3p) in NSCLC. In subsequent studies, it was determined that MSRB3, FCER1G and LTB4R2 genes, especially the HTR4 gene, could be potential target genes for lung cancer.