microRNA Sequencing Research Articles

Integrative RNA, miRNA, and 16S rRNA sequencing reveals immune-related regulation network for glycinin-induced enteritis in hybrid yellow catfish, Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂

Glycinin-induced foodborne enteritis is a significant obstacle that hinders the healthy development of the aquatic industry. Glycinin causes growth retardation and intestinal damage in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂), but its immune mechanisms are largely unknown. In the current study, five experimental diets containing 0% (CK), 1.74% (G2), 3.57% (G4), 5.45% (G6), and 7.27% (G8) immunological activity of glycinin were fed to juvenile hybrid yellow catfish to reveal the mechanism of the intestinal immune response to glycinin through RNA and microRNA (miRNA) sequencing and to explore the interrelation between immune molecules and intestinal microbiota. The results demonstrated that glycinin content in the posterior intestine increased significantly and linearly with the rise of dietary glycinin levels. More than 5.45% of dietary glycinin significantly reduced the nutritional digestion and absorption function of the posterior intestine. Notably, an obvious alteration in the expression levels of inflammatory genes (tnf-α, il-1β, il-15, and tgf-β1) of the posterior intestine was observed when dietary glycinin exceeded 3.57%. Sequencing results of RNA and miRNA deciphered 4,246 differentially expressed genes (DEGs) and 28 differentially expressed miRNAs (DEmiRNAs) between the CK and G6 groups. Furthermore, enrichment analysis of DEGs and DEmiRNA target genes exhibited significant responses of the MAPK, NF-κB, and WNT pathways following experimental fish exposure to 5.45% dietary glycinin. Additionally, at the level of 3.57% in the diet, glycinin obviously inhibited the increase of microbiota, especially potential probiotics such as Ruminococcus bromii, Bacteroides plebeius, Faecalibacterium prausnitzii, and Clostridium clostridioforme. In sum, 5.45% dietary glycinin through the MAPK/NF-κB/WNT pathway induces enteritis, and inflammatory conditions could disrupt micro-ecological equilibrium through miRNA secreted by the host in hybrid yellow catfish. This study constitutes a comprehensive transcriptional perspective of how intestinal immunity responds to excessive glycinin in fish intestines.

MicroRNA-mediated Ets1 repression in retinal endothelial cells: A novel anti-angiogenic mechanism in nonproliferative diabetic retinopathy.

This study aimed to discover the regulatory mechanisms contributing to angiogenesis in nonproliferative diabetic retinopathy (NPDR). This study employed a case-control design involving type 2 diabetes patients with and without NPDR. We utilised microRNA sequencing to analyse plasma and retina samples from T2D patients, to identify both existing and novel microRNAs relevant to retinal health. An integrative approach combining single-cell sequencing data from mouse and rat models was used to explore the molecular mechanism in retinal cells under diabetes condition. We identified a specific set of circulating microRNAs with strong predictive potential for distinguishing NPDR patients. In addition, a novel microRNA targeting the ETS proto-oncogene 1 (Ets1), a key regulator of microvascular angiogenesis, was found to be upregulated in the plasma of NPDR patients. Analysis of single-cell sequencing data suggested that Ets1 expression was downregulated in diabetic endothelial cells and was associated with the repression of Angiopoietin-1 and phosphoinositide 3-kinase-Akt (PI3K-Akt) signalling pathways, indicating an anti-angiogenic mechanism in NPDR. The identification of a novel microRNA involved in the anti-angiogenic mechanism in NPDR provides new insights into the molecular underpinnings of endothelial dysfunction in diabetic retinopathy. Our retina-specific circulating microRNA panel has potential utility in risk assessment and early detection of NPDR.

MicroRNA in pediatric pulmonary hypertension microRNA profiling to inform disease classification, severity, and treatment response in pediatric pulmonary hypertension.

Pediatric pulmonary hypertension is a heterogeneous disease associated with significant morbidity and mortality. MicroRNAs have been implicated as both pathologic drivers of disease and potential therapeutic targets in pediatric pulmonary hypertension. We sought to characterize the circulating microRNA profiles of a diverse array of pediatric patients with pulmonary hypertension using high-throughput sequencing technology. Peripheral blood samples were drawn from patients recruited at the time of a clinically indicated cardiac catheterization, and microRNA sequencing followed by differential expression and target/pathway enrichment analyses were performed. Among 63 pediatric patients with pulmonary hypertension, we identified specific microRNA signatures that uniquely classified patients by disease subtype, correlated with indicators of disease severity including invasive hemodynamic metrics, and changed over the course of treatment for pulmonary hypertension. These microRNA profiles include a number of specific microRNA molecules known to function in signaling pathways critical to pulmonary vascular biology and disease, including transforming growth factor-β (TGF-β), VEGF, PI3K/Akt, cGMP-PKG, and HIF-1 signaling. Circulating levels of miR-122-5p, miR-124-3p, miR-204-5p, and miR-9-5p decreased over the course of treatment in a subset of patients who had multiple samples drawn during the study period. Our findings support the further investigation of specific microRNAs as mechanistic mediators, biomarkers, and therapeutic targets in pulmonary hypertension.NEW & NOTEWORTHY We present novel insight into the circulating microRNA profiles of pediatric patients with pulmonary hypertension. Our findings support the utility of microRNAs as both useful biomarkers of disease severity and potential therapeutic targets in pediatric pulmonary hypertension.

Gestational diabetes mellitus-derived miR-7-19488 targets PIK3R2 mRNA to stimulate the abnormal development and maturation of offspring-islets.

Gestational diabetes mellitus (GDM) provides offspring with a hyper-metabolic intrauterine microenvironment. In this study, we aimed to identify key differential microRNAs in GDM-derived exosomes and explore the potential mechanisms of abnormal embryonic development of islets in offspring. Exosomes were extracted from umbilical vein blood of GDM and non-GDM (NGDM) parturients for microRNA sequencing. Offspring islets were collected on E18.5 and P0 to detect the expression and location of key proteins by immunofluorescence. Target binding of miR-7-19488 and PIK3R2 mRNA was verified using a dual-luciferase reporter assay. The miR-7-19488-mimic, PI3K/mTOR inhibitors were used to treat primarily islet cells to explore the relationship among miR-7-19488, PI3K, and Akt-FoxO1/mTORC1 signaling. The miR-7-19488 agomir was synthesized for further in vivo validation. GDM-derived exosomes caused the overdevelopment of offspring-islets at E18.5 with an increased production of insulin and glucagon co-staining cells, increased number of α cells synthesizing GLP-1, and stimulation of mTORC1 singling, which were more serious at birth. The up-regulated miR-7-19488 in GDM-exosomes targeted PIK3R2 mRNA, leading to translation stagnation of p85β and activation of PI3K-Akt singling in fetal islets. Importantly, the activated PI3K-Akt-FoxO1 singling promoted development and differentiation of α and β cells and enhanced the GLP-1/GLP-1R axis, which cooperates with miR-7-19488 to activate PI3K-Akt-FoxO1/mTORC1 singling, leading to the early initiation of the functional maturation of overdeveloped β cells. miR-7-19488 loaded in GDM-derived exosomes induce the abnormal overdevelopment and functional maturation of fetal islets, which is one of the contributors of high incidence of diabetes in adulthood.

A Practical Guideline for MicroRNA Sequencing Data Analysis in Chronic Lymphocytic Leukemia.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. They have been associated with several diseases and cancers, including chronic lymphocytic leukemia (CLL). CLL is the most common form of adult leukemia, and its pathogenesis is driven by the deletion of miRNAs, such as the miR-15a/16-1 cluster. In addition to initiating the development of CLL, the function of miRNAs in regulating the progression of this tumor remains to be investigated. Here, we present a computational pipeline, from the processing of miRNA sequencing files to functional analysis, including differential gene expression and gene set enrichment analysis.We exemplified the utility of the pipeline by applying it to genome-wide small RNA sequencing data from a cohort of CLL patients. The analysis revealed dysregulated expression profiles of miRNAs in CLL. The target genes of these miRNAs are not only associated with the response of CLL patients to current therapies but also involved in several cancer hallmarks, including the avoidance of cell death, the deregulation of cellular energetics, the activation of invasion and metastasis, and genome instability. The identified miRNA-gene interactions offer valuable insights for developing targeted therapies for CLL. In addition, we underscored the importance of a practical and robust computational pipeline to ensure the reliability and reproducibility of miRNA sequencing data analysis.

Pharmacogenetic and microRNA mechanisms of beta blocker use on bone.

Motivated by studies showing an association between beta blocker (BB) use and positive bone outcomes, a pilot randomized control trial (RCT) was performed at the Mayo Clinic which randomized postmenopausal women to placebo, propranolol (40 or 80 mg twice daily), atenolol (50 mg/day), or nebivolol (5 mg/day) to determine changes in bone turnover markers (BTMs) and in bone mineral density (BMD) over 20 weeks. Pharmacogenetic effects and microRNA-mediated mechanisms involving beta adrenergic receptor and related genes have previously been found. We sought to validate these effects and discover new candidates in an ancillary study to the pilot clinical trial. We genotyped all participants and performed microRNA (miRNA) sequencing at baseline and at 20 weeks for 24 participants from the atenolol or placebo groups. We discovered several variants in ADRB1, ADRB2, and HDAC4 which showed significant pharmacogenetic effects with BMD at multiple sites and with BTMs. Our miRNA results showed a significant treatment effect for miR-19a-3p over time with atenolol use in the low-responder group compared to placebo. Overall, the longitudinal miRNA analysis showed a large number of miRNAs which were up-regulated over the trial in the low responders but not the high responders compared to placebo, of which miR-19a-3p was one example. Finally, we compared the response to atenolol treatment for cardiovascular traits (pulse, blood pressure) with the response for the bone resorption marker, CTX, and found a largely independent effect. Our results have implications for personalized therapy and for understanding mechanisms of BB treatment effect on bone.

FRK exerts oncogenic effects by targeting NQO2 via exosomal miR-9-3p to regulate mitochondrial function.

The role of Fyn-related kinase (FRK) in promoting tumor progression and metabolism in non-small cell lung cancer (NSCLC) has been demonstrated in our earlier study, and here we further explored whether exosome could serve as a key player in the relevant regulatory mechanism. Exosomes were isolated from the culture medium of FRK-knockout (KO) cells and co-cultured with NSCLC cells, showing negative effects on cell proliferation, metastasis, metabolism, and mitochondrial function. Exosomal miR-9-3p was selected as an intermediate messenger through microRNA (miRNA) sequencing. The miR-9-3p inhibitor and exosome inhibitor GW4869 were then applied in the co-culture system to verify the contribution of exosomal miR-9-3p to cell malignant phenotype and mitochondrial function. Moreover, NQO2, a potential target of exosomal miR-9-3p, was also suggested to be involved in the regulation of mitochondrial functional status. In brief, this study revealed a novel molecular mechanism by which FRK promotes the malignant progression of NSCLC by targeting NQO2 via exosomal miR-9-3p to strengthen mitochondrial function.

Comprehensive bioinformatics analysis of metabolism‑related microRNAs in high myopia in young and old adults with age‑related cataracts.

High myopia and age‑related cataracts are prevalent ocular disorders that compromise visual acuity. The molecular mechanisms underlying these conditions remain largely unclear. Here, microRNA (miRNA or miR) sequencing was performed on aqueous humor samples obtained from individuals with age‑related cataracts and high myopia (AH, n=9), young patients with high myopia (YH, n=9) and a control group of elderly patients with age‑related cataracts, matched in terms of sex and age (AN, n=9). miRNA sequencing and differential expression were performed. Intersecting miRNAs were identified, as well as metabolism‑related genes from MsigDB were intersected with miRNA target genes. Functional enrichment was performed and disease targets predicted using DisGeNET. A protein‑protein interaction network was built with STRING, and hub genes were identified via Cytoscape. GeneMANIA analyzed hub genes, while drug predictions were made using Comparative Toxicogenomics Database. Long non‑coding RNAs and transcription factors were predicted via mirNet and ChEA3. Results were validated by RT‑qPCR. A total of 18 miRNAs were significantly differential expressed between AH and AN group, of which eight were up‑ and 10 were downregulated. A total of 23 miRNAs were significantly differential expressed between the YH and AN group, of which six were up‑ and 17 were downregulated. hsa‑miR‑490‑3p, hsa‑miR‑4423‑3p and hsa‑miR‑4485‑3p may serve as characteristic miRNAs. A total of 289 target genes were predicted. Functional enrichment analysis yielded 169 terms, with 'herpes simplex virus 1 infection' the most significantly enriched. There were 19 metabolism‑associated target genes linked with these miRNAs, suggesting a potential role of metabolic processes in pathogenesis of these conditions. The biosynthetic process of carbohydrate derivatives may serve a key role during the development of high myopia. There were 10 hub genes and Propionyl‑CoA Carboxylase Subunit β could potentially serve as a biomarker. Drugs that could modulate their function were predicted; cyclosporine, tretinoin and acetaminophen may exert a broad influence on these hub genes. Hub gene networks based on the miRNAs were constructed to predict 44 associated long non‑coding RNAs and 98 transcription factors. The present findings offer novel insights into the molecular mechanisms of age‑related cataracts and high myopia and propose potential therapeutic targets.

Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies.

Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy. We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C. Transduction, transgene expression, and biological activity were characterized in human retinal cells in vitro and in NHPs. R100 demonstrated superior retinal cell transduction in vitro and in vivo compared to AAV2, a commonly used wild-type AAV serotype in retinal gene therapies. Transduction of human retinal pigment epithelial cells in vitro by 4D-150 resulted in dose-dependent transgene expression and corresponding reductions in VEGF-A and VEGF-C. Intravitreal administration of 4D-150 to NHPs was well tolerated and led to robust retinal expression of both transgenes. In a primate model of laser-induced choroidal neovascularization, 4D-150 completely prevented clinically relevant angiogenic lesions at all tested doses. These findings support further development of 4D-150. Clinical trials are underway to establish the safety and efficacy of 4D-150 in individuals with wet AMD and DME.

The mechanism of exosomes of BMSCs modified with Bu Shen Yi Sui capsule in promoting remyelination via regulating miR-15b/Wnt signaling pathway-mediated differentiation of oligodendrocytes.

The Bu Shen Yi Sui capsule (BSYS), a modified version of the classical Chinese medicine formula Liu Wei Di Huang pill, has demonstrated therapeutic efficacy in the treatment of multiple sclerosis (MS). Nevertheless, the precise mechanism through which BSYS facilitates remyelination remains to be elucidated. This research investigates the role and potential mechanisms of BSYS-modified exosomes (exos) derived from bone marrow mesenchymal stem cells (BMSCs) in promoting remyelination in a cuprizone (CPZ)-induced demyelination model in mice. C57BL/6 mice were administered a 0.2% CPZ-containing diet for 5 weeks to induce demyelination, followed by treatment with exosomes derived from BMSC (BMSC-exos) and BSYS-modified BMSC exosomes (BSYS-BMSC-exos) twice weekly for 2 weeks. Body weight measurements were recorded, and motor function was evaluated using the rotarod test. Pathological changes in myelin and axons were assessed via Luxol fast blue (LFB) staining, transmission electron microscopy (TEM), and immunofluorescence (IF) staining. Oligodendrocyte proliferation, differentiation, and maturation were analyzed using double immunofluorescence staining, Western blot (WB), and real-time quantitative reverse transcription PCR (qRT-PCR). Additionally, microRNA (miRNA) sequencing and a luciferase reporter assay were conducted to verify miRNA binding to its target gene. Key markers of the Wnt/β-catenin signaling pathway were examined using WB and qRT-PCR. BSYS-BMSC-exos treatment significantly increased both body weight and rotarod performance in CPZ mice. Moreover, BMSC-exos and BSYS-BMSC-exos reversed myelin loss and axonal damage. These treatments enhanced oligodendrocyte proliferation, differentiation, and maturation, with BSYS-BMSC-exos exhibiting a particularly pronounced effect on the expression of adenomatous polyposis coli clone CC1 (CC1), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP). Sequencing and luciferase assays revealed that miR-15b-5p, enriched in BSYS-BMSC-exos, directly targets Wnt3a. Furthermore, BSYS-BMSC-exos elevated axis inhibition protein 2 (Axin2) expression while markedly reducing Wnt family member 3A (Wnt3a), phospho-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and T-cell specific transcription factor 4/transcription factor 7-like 2 (TCF4/TCF7L2) levels. The findings suggest that BSYS-BMSC-exos alleviate neurological deficits, enhance oligodendrocyte differentiation and maturation, and promote remyelination in CPZ mice. miR-15b-5p, enriched in BSYS-BMSC-exos, targets and downregulates Wnt3a, thereby inhibiting the Wnt/β-catenin signaling pathway.

Macrophage membrane-encapsulated miRNA nanodelivery system for the treatment of hemophilic arthritis

Hemophilic arthritis (HA) is one of the most pathologically altered joint diseases. Specifically, periodic spontaneous hemorrhage-induced hyperinflammation of the synovium and irreversible destruction of the cartilage are the main mechanisms that profoundly affect the behavioral functioning and quality of life of patients. In this study, we isolated and characterized platelet-rich plasma-derived exosomes (PRP-exo). We performed microRNA (miRNA) sequencing and bioinformatics analysis on these exosomes to identify the most abundant miRNA, miR-451a. Following this, we developed an M@ZIF-8@miR nanotherapeutic system that utilizes nanoscale zeolitic imidazolate framework (ZIF) as a carrier for miRNA delivery, encapsulated within M2 membranes to enhance its anti-inflammatory effects. In vitro and in vivo studies demonstrated that M@ZIF-8@miR significantly reduced pro-inflammatory cytokines, controlled synovial inflammation, and achieved potent therapeutic efficacy by reducing joint damage. We suggest that the ability of M@ZIF-8@miR nanocomposites to inhibit pro-inflammatory cytokines, enhance cellular uptake, and exhibit good endosomal escape properties makes them promising carriers for the efficient delivery of therapeutic nucleic acid drugs. This approach delays joint degeneration and provides a promising combinatorial strategy for HA treatment.

Human liver progenitor-like cells-derived extracellular vesicles promote liver regeneration during acute liver failure

Hepatocyte-derived liver progenitor-like cells (HepLPCs) exhibit a remarkable capacity to support liver function by detoxifying ammonia, promoting native liver regeneration, and suppressing inflammation, which leads to improvements in the recovery and survival of animals with acute liver failure (ALF). However, the mechanism through which HepLPCs promote liver regeneration is unclear. Here, we isolated HepLPC-derived extracellular vesicles (HepLPC-EVs) from conditioned media and performed microRNA sequencing analysis. Our results showed HepLPC-EVs promoted liver regeneration in mice with carbon tetrachloride or acetaminophen induced ALF. Cell cycle progression and proliferation of primary human hepatocytes were promoted after coculture with HepLPC-EVs. Exosomal miRNA sequencing confirmed that HepLPC-EVs were enriched with miR-183-5p, which played an essential role in ameliorating ALF. Mechanistically, HepLPC-derived exosomal miR-183-5p negatively regulated the expression of the target gene FoxO1, activated the Akt/GSK3β/β-catenin signaling pathway, and thereby promoted liver regeneration and restoration of normal liver function. These results indicate that during ALF, HepLPC-Exos mediate liver regeneration mainly through a paracrine exosome-dependent mechanism and these effects accelerate liver regeneration and lead to the restoration of normal liver function.Graphical

The Intra-Articular Delivery of a Low-Dose Adeno-Associated Virus-IL-1 Receptor Antagonist Vector Alleviates the Progress of Arthritis in an Osteoarthritis Rat Model.

Background/Objectives: Interleukin-1 (IL-1) is a pivotal mediator in the pathological progression of osteoarthritis (OA), playing a central role in disease progression. However, the rapid clearance of IL-1 receptor antagonist (IL-1Ra) from the joints may hinder the efficacy of intra-articular IL-1Ra injections in reducing OA-associated pain or cartilage degradation. Methods: Sustaining sufficient levels of IL-1Ra within the joints via adeno-associated virus (AAV)-mediated gene therapy presents a promising therapeutic strategy for OA. In this study, we constructed an IL-1Ra expression cassette employing intron insertion in the coding sequence (CDS) region to enhance protein expression levels. Furthermore, we incorporated precisely targeted liver-specific microRNA (miRNA) sequences to specifically downregulate transgene expression within hepatic tissues, thereby ensuring more targeted and controlled regulation of gene expression. Results: A rat model of OA was employed to compare the efficacy of AAV5 and AAV9 for IL-1Ra delivery at both high and low doses. It was observed that low-dose, but not high-dose, AAV9-IL-1Ra resulted in a significant reduction in joint swelling, accompanied by a decrease in the diameter of the affected area and the preservation of biomarkers associated with trabecular bone integrity. Conclusions: These results highlight the great potential of AAV9-IL-1Ra in osteoarthritis therapy, with the promise of achieving long-term improvement through a single intra-articular injection.

Abstract A008: Identification of EYA2 as a promising biomarker for DICER1-related tumor predisposition

Abstract Background: DICER1-related tumor predisposition (DRTP), also known as DICER1 syndrome, is a genetic condition associated with an increased risk of developing various tumors, including pleuropulmonary blastoma, thyroid gland neoplasia, ovarian tumors, and cystic nephroma. Currently, the diagnosis and surveillance of DRTP primarily relies on genetic testing for DICER1 mutations. There is a pressing need for surrogate diagnostic biomarkers to improve early detection and monitoring. Objective: To identify and validate EYA2 as a potential biomarker for DICER1-related tumor predisposition. Methods: Murine mesenchymal stromal cell lines were developed to model the consequences of DICER1 mutations, including the DICER1E1705K mutation found in DRTP. Both bulk RNA sequencing (RNAseq) and microRNA sequencing (miRNAseq) were employed to identify differentially expressed mRNAs in these cell lines. The RNA and protein levels of Eya2 were measured by qRT-PCR and Western blot, respectively. De-repression of Eya2 mRNA by miRNA was evaluated by dual luciferase assay to link overexpression with the DICER1 defect of DRTP. Then, immunohistochemistry staining of EYA2 was performed on a panel of DRTP tumors. Results: We found that more than 2000 mRNAs were derepressed in mesenchymal stromal cells expressing DICER1E1705K as compared to DICER1WT. Among these, Eya2 emerged as a top candidate. We observed elevated Eya2 mRNA and protein levels in DICER1E1705K -expressing mesenchymal stromal cells. Utilizing a reporter assay, we also observed de-repression of the 3’ UTR of Eya2 upon expression of DICER1E1705K as compared to DICER1WT. Strong nuclear EYA2 staining was observed in a subset of DRTP tumor samples. Conclusion: Elevated EYA2 expression in tumor tissues from DRTP patients supports its potential use in both diagnostic and surveillance contexts. Further studies are warranted to validate EYA2 as a clinical biomarker and explore its utility in improving early detection and monitoring strategies for individuals at risk of DRTP. Citation Format: Mona Wu, Shengmei Zhou, Felix Kommoss, Yemin Wang, James F Amatruda. Identification of EYA2 as a promising biomarker for DICER1-related tumor predisposition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A008.

Alterations in the Levels of Urinary Exosomal MicroRNA-183-5p and MicroRNA-125a-5p in Individuals with Type 2 Diabetes Mellitus.

Background: Type 2 diabetes mellitus (T2DM) is a metabolic disorder, and urinary exosomal microRNAs (miRNAs) were utilized as potential disease prediction or diagnostic biomarkers in numerous studies. This study investigated the differential expression of urinary exosomal miRNAs between non-diabetes mellitus (NDM) individuals and those with T2DM. Aim: To elucidate the association between urinary exosomal miRNAs and T2DM. Methods: We recruited patients diagnosed with T2DM and NDM individuals in West China Hospital, Sichuan University, from November 2023 to February 2024. Subsequently, we performed sequencing of urinary exosomal microRNAs in both groups. The obtained sequencing results were further validated using RT-qPCR in both the training set and the validation set. Additionally, we conducted logistic regression analysis and Spearman correlation analysis on miRNAs with significant differential expression, as well as analysis of their biological functions. Results: A total of 118 urine samples were collected, 59 from individuals diagnosed with T2DM and 59 from NDM. There were differentially expressed miR-183-5p (p = 0.034) and miR-125a-5p (p = 0.008) between the two groups. Furthermore, multivariate regression analysis demonstrated that higher miR-125a-5p levels were negatively associated with the risk of T2DM (p = 0.044; OR: 0.046; 95% CI: 0.002, 0.922). Bioinformatics analysis indicated that the target genes of miR-183-5p were predominantly involved in insulin signaling and glucose transport processes, while those target genes of miR-125a-5p primarily mediated autophagy. Conclusions: miR-183-5p and miR-125a-5p might be involved in the pathogenesis of T2DM, while higher urinary exosomal miR-125a-5p was negatively associated with the risk of T2DM.

MicroRNAome profiling of breast cancer unveils hsa-miR-5683 as a tumor suppressor microRNA predicting favorable clinical outcome

BackgroundBreast cancer is a heterogeneous disease with diverse molecular subtypes, underscoring a better understanding of its molecular features and underlying regulatory mechanisms. Therefore, identifying novel prognostic biomarkers and therapeutic targets is crucial for advancing the current standard of care for breast cancer patients.MethodsNinety-six formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent miRNAome profiling using QIAseq microRNA library kit and sequencing on Illumina platform. Mature miRNA quantification was conducted using CLC Genomics Workbench v21.0.5, while Relapse-free survival (RFS) analysis was conducted using RStudio 2023.09.1. Gain-of-function studies were conducted using miRNA mimics, while the effects of miRNA exogenous expression on cancer hallmark were assessed using 2-dimentional (2D) proliferation assay, three-dimensional (3D) organotypic culture, and live-dead staining. TargetScan database and Ingenuity Pathway Analysis (IPA) were used for miRNA target identification.ResultsHierarchical clustering based on miRNA expression revealed distinct patterns in relation to PAM50 classification and identified miRNAs panels associated with luminal, HER2, and basal subtypes. hsa-miR-5683 emerged as a potential prognostic biomarker, showing a favorable correlation with RFS and suppressing tumorigenicity under 2D and 3D conditions in triple-negative breast cancer (TNBC) models. Findings were further extended to the MCF7 hormone receptor positive (HR+) model. Transcriptomic profiling of hsa-miR-5683 overexpressing TNBC cells revealed its potential role in key oncogenic pathways. Integration of downregulated genes and CRISPR-Cas9 perturbational effects identified ACLY, RACGAP1, AK4, MRPL51, CYB5B, MKRN1, TMEM230, NUP54, ANAPC13, PGAM1, and SOD1 as bona fide gene targets for hsa-miR-5683.ConclusionsOur data provides comprehensive miRNA expression atlas in breast cancer subtypes and underscores the prognostic and therapeutic significance of numerous miRNAs, including hsa-miR-5683 in TNBC. The identified gene targets unravel the intricate regulatory network in TNBC progression, suggesting promising avenues for further research and targeted therapeutic interventions.

BIOM-76. EXPLORING CSF MICRORNA SIGNATURES AS DIAGNOSTIC BIOMARKERS IN IDH-WILDTYPE DIFFUSE GLIOMAS

Abstract The most prevalent malignant tumors of the central nervous system are adult-type diffuse gliomas. Isocitrate dehydrogenase (IDH) mutations are identified in a subset of diffuse gliomas, leading to two distinct subtypes of gliomas: IDH-wildtype and IDH-mutant. In comparison to IDH-mutant gliomas, IDH-wildtype gliomas tend to be associated with a more rapid progression and lower overall survival rates. The analysis of differentially expressed microRNAs (DE-miRNAs) in cerebrospinal fluid (CSF) of IDH-wildtype diffuse gliomas could provide an effective and non-invasive approach to assist in the early diagnosis, classification, and management of the disease. Extracellular vesicle (EV)-derived miRNAs obtained from the CSF of 11 patients with IDH-wildtype diffuse gliomas and 8 controls were analyzed by microRNA sequencing (miRNA-seq). DE-miRNAs were defined using p-value≤0.05 and absolute log2 fold change (log2FC) ≥1. Candidate DE-miRNAs were validated by qRT-PCR in 24 samples (15 IDH-wildtype vs. 9 control) using the 2-ΔΔCT method, followed by receiver operating characteristic (ROC) curve analysis. Hsa-miR-24-3p was used as the internal control. miRNA-seq analysis identified hsa-miR-484, hsa-miR-223-3p, hsa-miR-1246, hsa-miR-323a-3p, hsa-miR-106b-3p, hsa-miR-21-5p, hsa-miR-15a-5p, has-miR-15b-5p, and hsa-miR-92a-3p among the top 20 upregulated miRNAs in the CSF of patients with IDH-wildtype diffuse gliomas. Further analysis by qPCR showed that patients with IDH-wildtype diffuse gliomas had significantly higher levels of has-miR-21-5p (2.28 ± 2.33) compared to control subjects (-2.22e-007 ± 1.08, p=0.012). The ROC curve analysis revealed an area under the curve (AUC) of 0.84 (95% CI, 0.68-1.0) for has-miR-21-5p CSF levels in distinguishing IDH-wildtype diffuse gliomas from control subjects. EV-derived miRNAs in CSF, including has-miR-21-5p, have the potential to distinguish IDH-wildtype diffuse gliomas from control individuals in a minimally-invasive manner and serve as diagnostic biomarkers.

MiR-199a-5p aggravates renal ischemia-reperfusion and transplant injury by targeting AKAP1 to disrupt mitochondrial dynamics.

Renal ischemia-reperfusion injury (IRI) is a complex pathophysiological process and a major cause of delayed graft function (DGF) after transplantation. MicroRNA (miRNA) has important roles in the pathogenesis of IRI and may represent promising therapeutic targets for mitigating renal IRI. miRNA sequencing was performed to profile microRNA expression in mouse kidneys after cold storage and transplantation (CST). Lentivirus incorporating a miR-199a-5p modulator was injected into mouse kidney in situ before syngenetic transplantation and unilateral IRI to determine the effect of miR-199a-5p in vivo. miR-199a-5p mimic or inhibitor was transfected cultured tubular cells before ATP depletion recovery treatment to examine the role of miR-199a-5p in vitro. Sequencing data and microarray showed upregulation of miR-199a-5p in mice CST and human DGF samples. Lentivirus incorporating a miR-199a-5p mimic aggravated renal IRI, and protective effects were obtained with a miR-199a-5p inhibitor. Treatment with the miR-199a-5p inhibitor ameliorated graft function loss, tubular injury, and immune response after CST. In vitro experiments revealed exacerbation of mitochondria dysfunction upon ATP depletion and repletion model in the presence of the miR-199a-5p mimic, whereas dysfunction was attenuated when the miR-199a-5p inhibitor was applied. miR-199a-5p was shown to target A-kinase anchoring protein 1 (AKAP1) by double luciferase assay and miR-199a-5p activation reduced dynamin-related protein 1 (Drp1)-s637 phosphorylation and mitochondrial length. Overexpression of AKAP1 preserved Drp1-s637 phosphorylation and reduced mitochondrial fission. miR-199a-5p activation reduced AKAP1 expression, promoted Drp1-s637 dephosphorylation, aggravated the disruption of mitochondrial dynamics, and contributed to renal IRI.NEW & NOTEWORTHY This study identifies miR-199a-5p as a key regulator in renal ischemia-reperfusion injury through microRNA sequencing in mouse models and human delayed graft function. miR-199a-5p worsens renal IRI by aggravating graft dysfunction, tubular injury, and immune response, while its inhibition shows protective effects. miR-199a-5p downregulates A-kinase anchoring protein 1 (AKAP1), reducing dynamin-related protein 1 (Drp1)-s637 phosphorylation, increasing mitochondrial fission, and causing dysfunction. Targeting the miR-199a-5p/AKAP1/Drp1 axis offers therapeutic potential for renal IRI, as AKAP1 overexpression preserves mitochondrial integrity by maintaining Drp1-s637 phosphorylation.

Huangqi-Danshen decoction improves heart failure by regulating pericardial adipose tissue derived extracellular vesicular miR-27a-3p to activate AMPKα2 mediated mitophagy

BackgroundHuangqi-Danshen decoction (HDD) is a classic traditional Chinese medicine for treating heart failure. Pericardial adipose tissue (PAT) has recently gained increasing attention in cardiovascular diseases. PurposeThis study aimed to investigate the effect of pericardial adipose tissue-derived extracellular vesicles on heart failure, the protective effect of HDD on myocardial remodel in heart failure rats, and identify the potential molecular mechanisms involved. MethodsUPLC-MS/MS identified active components of HDD. Extracellular vesicles (EVs) from pericardial adipose tissue of sham-operated and HF rats were identified through transmission electron microscopy, nanoparticle tracking analysis and western blot. EVs were co-cultured with H9c2 cardiomyocytes in order to examine their uptake and effects. MicroRNA sequencing, dual-luciferase reporter assay and PCR were conducted for exploring specific mechanisms of EVs on hypertrophic cardiomyocytes. In vivo, heart failure was modeled in rats via transverse aortic constriction (TAC). In vitro, the hypertrophic cardiomyocyte model were established using Ang II-induced H9c2 cardiomyocytes. ResultsUPLC-MS/MS identified 11 active components in serum of HDD administrated rats. Echocardiography showed HDD improved cardiac function in TAC model rats. HE and Masson staining indicated HDD ameliorated myocardial hypertrophy and fibrosis. MicroRNA sequencing found that HDD treatment resulted in 37 differentially expressed miRNAs (DMEs) (p < 0.05 and |log2FC| ≥ 1). KEGG analysis revealed that DEMs were enriched in the AMPK signaling pathway. PCR identified miR-27a-3p with the greatest difference in AMPK-related DMEs. Dual-luciferase reporter assay and Targetscan website were utilized to identify the target relationship between miR-27a-3p and PRKAA2 (AMPKα2). The miR-27a-3p negatively regulated AMPKα2 to inhibit mitophagy mediated by PINK1/Parkin pathway. HDD inhibited miR-27a-3p secretion from failing heart pericardial adipose tissue-derived extracellular vesicles, thereby improving inflammation, cardiac function, and myocardial remodeling through above pathways. ConclusionHDD inhibited the PAT-derived extracellular vesicular miR-27a-3p in failing hearts to activate AMPK/PINK1/Parkin signaling-mediated mitophagy, which improved cardiomyocyte energy metabolism, myocardial remodeling and heart failure.

Identification of Key miRNAs in Endometriosis.

Endometriosis, a prevalent gynecological disorder characterized by the presence of endometrial-like tissue outside the uterus, poses significant challenges in diagnosis and management due to its unclear pathogenesis and lack of specific biomarkers. This study investigates the potential use of microRNAs (miRNAs) as key markers in endometriosis by studying two cohorts of patients (14 patients diagnosed with endometriosis and 15 patients with gynecological benign lesions, different from endometriosis). MicroRNA sequencing analysis was tested within data management by a custom pipeline designed by Eurofins Genoma Group. We identified a specific miRNA expression profile associated with endometriosis to feature specific disease molecular clusters to further elucidate the underlying mechanisms driving endometriosis pathogenesis. Data from the present study suggest a specific miRNA scar for endometriosis compared to other gynecological diseases to develop screening tools in early diagnosis and to ameliorate the management of the disease itself. This study lays the foundation for the identification of key miRNAs involved in the disease pathogenesis to unveil the molecular signatures in the complex scenario of endometriosis. Further validation and exploration of these findings are needed to develop tools to improve molecular diagnosis and to create a machine-learning prediction algorithm in the future.