Exosomal miR-21 Research Articles

Concanavalin A-activated magnetic nanoparticles as an affine material for urinary exosome isolation.

Exosomes are a type of membrane vesicle secreted into the extracellular medium by most cell types. They have a great potential for clinical practice as noninvasive biomarkers for diagnosis of various diseases, prognosis, and monitoring of therapy, which stimulates the development of simple methods for isolating exosomes from biological fluids. A novel affine material based on aminosilanized superparamagnetic core‒shell nanoparticles for fast isolation of urinary exosomes is reported. Iron oxide nanoparticles coated with amino organosilane have been synthesized. The structural and magnetic characteristics of the resulting nanoparticles have been studied by transmission electron microscopy and ferromagnetic resonance. The surface of the synthesized nanoparticles has been chemically functionalized with lectin (concanavalin A), and the efficiency of the obtained material as a sorbent for affine exosome isolation from human urine has been demonstrated. A highly purified fraction of exosomes 90-200nm in size has been obtained. The exosomal nature of the isolated vesicles has been confirmed by bioluminescent solid-phase microassay of tetrasporine receptor markers. The presence of exosomal miR-21 in the isolated human urine exosome samples has been established.

Association of elevated exosomal miR-21 levels with nonunion in clavicular fractures post-ORIF: A prospective analysis

BackgroundThis study investigates the relationship between plasma exosomal miRNAs and nonunion risk following open reduction and internal fixation (ORIF) of clavicle fractures, aiming to identify predictive molecule to enhance patient management and personalized orthopedic care. MethodsConducted as a prospective cohort study at Taian City Central Hospital, participants included individuals with unilateral, closed clavicle fractures who underwent ORIF (n = 763, 546 males and 217 females). Plasma samples were collected preoperatively for exosome isolation and miRNA extraction, specifically analyzing miR-100, miR-124, miR-125b, and miR-21 using quantitative polymerase chain reaction (qPCR). Patients were classified into union and nonunion groups based on follow-up outcomes at a minimum of three months post-surgery. ResultsOut of 763 patients, 720 achieved union while 43 developed nonunion. Notably, the nonunion group exhibited significantly elevated exosomal miR-21 expression levels. Univariate analysis demonstrated a significant association between high miR-21 expression and nonunion occurrence (Relative Risk [RR] = 1.82, P = 0.0004). Multivariate logistic regression analysis corroborated this association (RR > 1.8, P < 0.05), adjusting for covariates. High miR-21 levels (3.12 to 7.89) were robustly associated with nonunion outcomes (RR > 4), independent of other factors. Receiver operating characteristic (ROC) curve analysis indicated certain clinical diagnostic value of miR-21 for predicting nonunion (Area Under the Curve [AUC] = 0.7885). ConclusionsThe elevated preoperative levels of exosomal miR-21 were significantly associated with an increased risk of bone nonunion at three months ORIF in patients with clavicle fractures, indicating that miR-21 holds potential as a non-specific predictive molecule.

Liquid biopsy for CNS lymphoma: CSF exosomes and CSF exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b are promising biomarkers for diagnosis.

Central nervous system lymphoma (CNSL) is a devastating disease with a poor prognosis. Early diagnosis, monitoring of the treatment response, and outcome prediction carry the utmost importance in the management of patients with CNSL. Surgical biopsy is the gold standard for tissue diagnosis, however, this procedure has potential complications. Therefore, there is a need for a method that provides information about diagnosis and patient monitoring to avoid surgical risks. The study aimed to investigate potential diagnostic biomarkers for patients with CNSL. Patients with secondary CNSL were included in this study. Serum and cerebrospinal fluid (CSF) samples were collected before treatment and after completion of the treatment. Cell-free DNA (cfDNA), exosomes, free and exosomal microRNA (miR)-15a, miR-21, miR-155, miR-210, and miR-19b in both serum and CSF were examined, and they were compared with the controls. Also, their levels before and after treatment were compared. Nine patients with the diagnosis of secondary CNSL were reviewed. cfDNA, miR-15a, and miR-155 in serum, and exosome in CSF were found to be significantly higher in CNSL patients compared to the controls. Exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b in CSF were found to be significantly higher in CNSL patients compared to controls, whereas their levels in serum were not significantly high. Our findings suggested that exosomes and exosomal miR-15a, miR-21, miR-155, miR-210 and miR-19b in CSF would be promising biomarkers for the diagnosis of patients with CNSL. Further studies are needed to confirm our findings.

Diagnostic and screening potential of plasma exosome miR‑99b‑5p and its combination with other miRNAs for colorectal cancer.

Extracellular vesicles (EVs) secreted by tumor cells have been documented to hold viable biomarker potential. Therefore, the present study evaluated the potential clinical value of EV-microRNAs (miRNAs or miRs) in the plasma exosomes of patients with colorectal cancer (CRC) for the early diagnosis and screening of CRC. In total, 95 plasma samples were collected at The Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) between 2017 and 2019. Specifically, 68 samples were from patients with CRC and 27 were from healthy control (HC) donors. High-throughput sequencing was used to detect the expression of miRNAs in the isolated plasma EVs, which was subsequently verified by reverse transcription-quantitative PCR. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic potential of single and combined miRNAs for CRC. Bioinformatics analysis was employed to predict the target genes of candidate miRNAs. Compared with those in the HC group, the CRC group expressed higher levels of miR-99b-5p and miR-409-3p, especially during the early stages of CRC. Clinicopathological analysis confirmed the higher expression levels of miR-99b-5p during the early stages, as well as higher expression levels in the colon compared with those in the rectum. ROC curve analysis revealed that the area under the curve (AUC) of miR-99b-5p for the diagnosis of early CRC was 73.5% (P=0.007). The early diagnostic capability of miR-99b-5p combined with miR-409-3p for CRC was evaluated, and the AUC was found to be 74.1% (P=0.006). In addition, the AUC of the combination of miR-99b-5p, miR-409-3p and carcinoembryonic antigen was 81.2% (P<0.001), indicating that this three-parameter combination displayed higher diagnostic power compared with any single miRNA for early CRC screening. The results from the present study suggest that the expression of miR-99b-5p in plasma exosomes is significantly upregulated in CRC, which holds potential for the early diagnosis of this cancer type. Such potential can be enhanced further by combining it with other miRNAs. Therefore, the present study provides a comprehensive but preliminary insight for the viability of miR-99b-5p (alone or combined with other miRNAs) for CRC diagnosis, which requires further exploration in the future.

A microfluidic-based chemiluminescence biosensor for sensitive multiplex detection of exosomal microRNAs based on hybridization chain reaction

The analysis of microRNAs (miRNAs) in exosomes is of great importance for noninvasive early disease diagnosis. However, current techniques to detect exosomal miRNAs is hampered either by laborious exosome isolation or low abundance of miRNAs in exosomes. Here, we developed a microfluidic chemiluminescence (CL) analysis method for the multiplexed detection of exosomal miR-21 and miR-155. The microfluidic device contained three parts: a snake-shaped channel for fully mixing chemiluminescent reagents, a ship-shaped channel modified with CD63 protein aptamer for capturing exosomes, and another two parallel ship-shaped channels for hybridization chain reaction (HCR) amplification and CL detection. The multiple signal amplification was realized by Y-shaped arrays, HCR amplification, and poly-HRP catalyzed CL reaction. Using this multiple signal amplification method, our microfluidic CL biosensor achieves a limit of detection of miRNAs of 0.49 fM, with a linear range of 1 fM–10 pM, which is better or comparable to previously reported biosensors. What's more, the proposed microfluidic biosensor exhibits great specificity and selectivity to the target miRNA. Moreover, the microfluidic CL strategy exhibited excellent accuracy and could significantly distinguish different cancer subtypes as well as cancer patients and healthy people. These results suggest that this simple, high sensitive, and more accurate analytical strategy by analyzing different types of exosomal miRNAs has the potential applications in cancer diagnosis and stage monitoring.

Expression of exosomal microRNAs miR-34a and miR-210 in male infertility: relationship with morphokinetic parameters and sperm DNA fragmentation

Introduction. Infertility affects tens of millions of men and women across the globe. In approximately half of cases, male factors are the cause of infertility. In recent decades, there has been a significant decline in the quality of male ejaculate, which is characterized by reduced sperm concentration and motility. The insufficient diagnostic and prognostic value of routine semen analysis results highlights the challenge of developing effective diagnostic tools and searching for reliable biomarkers of male infertility. One of the most promising approaches may be assessing sperm microRNA expression.Objective. To study the role of sperm exosomal microRNAs miR-34a and miR-210 in the development of male infertility.Materials &amp; methods. The retrospective study included 150 men aged 25 – 49 years; of these, 96 patients were diagnosed with idiopathic infertility. The comparison group consisted of 54 fertile men. To assess the structure and motility of sperm, the results of a standard ejaculate study (WHO, 2021) and computer data analysis using MMC Sperm (MMCSoft, St. Petersburg, Russia) software were used. The degree of DNA fragmentation was assessed using the TUNEL method. To analyze the expression of miR-34a and miR-210, quantitative real-time PCR was performed using the miRCURY LNA SYBR Green PCR Kit («Qiagen» GmbH, Hilden, Germany) and the Rotor-Gene Q PCR product detection system («Qiagen» GmbH, Hilden, Germany).Results. The study of ejaculate using the Computer Assisted Sperm Analysis System (CASA) method revealed a statistically significant relationship between the level of DNA fragmentation of sperm and indicators of the speed of their movement: rectilinear (VSL) (r = -0.522726; p &lt; 0.01), curvilinear (VCL) (r = -0.499096; p &lt; 0.01), along the middle path (VAP) (r = -0.429533; p &lt; 0.01), as well as with the amplitude of lateral head displacement (ALH) (r = -0.294779, p &lt; 0.01), the linearity of their curvilinear path (LIN) (r = -0.385796; p &lt; 0.01), the degree of straight-line movements (STR) (r = -0.268248; p &lt; 0.05) and their progressive mobility (r = -0.411547; p &lt; 0.01). A study of the level of microRNA expression in sperm exosomes revealed a statistically significant decrease in its miRNA-34a pool (p = 0.0116). According to the Chaddock scale, the strength of the correlation between miR-210 expression and the effectiveness of ART programs was moderate (0.437993). The inverse relationship between miR-34a expression and IVF and ICSI results was weak (0.135314).Conclusion. The analysis of exosomal microRNA-34a and microRNA-210, which are involved in the regulation of spermatogenesis, reveals a direct correlation between their variations and changes in the kinetic and morphological parameters of gametes. It also indicates a relationship with the state of DNA fragmentation. These findings suggest varying levels of gene expression among infertile patients, men with proven fertility, and those undergoing assisted reproductive technology (ART) treatments, both with successful and repeated unsuccessful outcomes.

The regulatory role of miR-21 in ferroptosis by targeting FTH1 and the contribution of microglia-derived miR-21 in exosomes to arsenic-induced neuronal ferroptosis

Arsenic is recognized as a hazardous environmental toxicant strongly associated with neurological damage, but the mechanism is ambiguous. Neuronal cell death is one of the mechanisms of arsenic-induced neurological injury. Ferroptosis is involved in the pathophysiological process of many neurological diseases, however, the role and regulatory mechanism of ferroptosis in nerve injury under arsenic exposure remains uncovered. Our findings confirmed the role of ferroptosis in arsenic-induced learning and memory disorder and revealed miR-21 played a regulatory role in neuronal ferroptosis. Further study discovered that miR-21 regulated neuronal ferroptosis by targeting at FTH1, a finding which has not been documented before. We also found an extra increase of ferroptosis in neuronal cells conditionally cultured by medium collected from arsenic-exposed microglial cells when compared with neuronal cells directly exposed to the same dose of arsenic. Moreover, microglia-derived exosomes removal or miR-21 knockdown in microglia inhibited neuronal ferroptosis, suggesting the role of intercellular communication in the promotion of neuronal ferroptosis. In summary, our findings highlighted the regulatory role of miR-21 in ferroptosis and the contribution of microglia-derived miR-21 in exosomes to arsenic-induced neuronal ferroptosis.

Abstract Tu001: MiR-21 mitigated pulmonary hypertension induced right ventricular dysfunction through pulmonary circulating exosomes

Background: Patients with pulmonary arterial hypertension (PAH) often experience adverse outcomes primarily due to right ventricular (RV) dysfunction. However, the intricate relationship between pulmonary circulation and RV remodeling remains largely unexplored. Exosomal microRNAs (miRNAs) function as paracrine signaling mediators in diverse diseases. Aims: To investigate the ability of exosomal miRNAs derived from pulmonary endothelial cells to transmit signals affecting cardiomyocytes. Methods and Results: Utilizing next-generation sequencing, we identified several target miRNAs by analyzing exosome samples obtained through right-heart catheterization. Notably, exosomal miR-21 exhibited the highest expression in the pulmonary circulation (Figure 1) . Under hypoxic conditions, the expression of miR-21 increased in human pulmonary endothelial cells (HPECs) and in exosomes derived from their conditioned medium. Treatment with these exosomes enhanced miR-21 expression and concurrently mitigated apoptosis in hypoxia-exposed cardiomyocytes. Likewise, miR-21 demonstrated a protective role against hypoxia-induced injuries and apoptosis, involving the SPRY-2/p-ERK and PTEN/Akt pathways. In a murine model of sugen/hypoxia-induced PAH, mice lacking miR-21 (miR-21 -/- ) exhibited more severe RV dysfunction, altered hemodynamics, and increased fibrosis compared to wild-type (WT) mice. Employing a parabiosis model, pairs consisting of WT and miR-21 -/- mice were shielded from RV dysfunction, whereas pairs of miR-21 -/- mice displayed exacerbated RV function (Figure 2) . Conclusion: Hypoxia stimulates the production of exosomal miR-21 in both HPECs and their microenvironment, subsequently mitigating apoptosis in cardiomyocytes (Figure 3) . Our findings propose endothelial cell-derived exosomal miR-21 as a promising therapeutic target for PAH.

Intermittent lysis on a single paper-based device to extract exosomal nucleic acid biomarkers from biological samples for downstream analysis.

As the role of exosomes in physiological and pathological processes has been properly perceived, harvesting them and their internal components is critical for subsequent applications. This study is a debut of intermittent lysis, which has been integrated into a simple and easy-to-operate procedure on a single paper-based device to extract exosomal nucleic acid biomarkers for downstream analysis. Exosomes from biological samples were captured by anti-CD63-modified papers before being intermittently lysed by high-temperature, short-time treatment with double-distilled water to release their internal components. Exosomal nucleic acids were finally adsorbed by sol-gel silica for downstream analysis. Empirical trials not only revealed that sporadically dropping 95°C ddH2O onto the anti-CD63-modified papers every 5min for 6 times optimized the exosomal nucleic acids extracted by the anti-CD63 paper but also verified that the whole deployed procedure is applicable for point-of-care testing (POCT) in low-resource areas and for both in vitro (culture media) and in vivo (plasma and chronic lesion) samples. Importantly, downstream analysis of exosomal miR-21 extracted by the paper-based procedure integrated with this novel technique discovered that the content of exosomal miR-21 in chronic lesions related to their stages and the levels of exosomal carcinoembryonic antigen originated from colorectal cancer cells correlated to their exosomal miR-21.

M1 Macrophage-Derived Exosomal MiR-155 Enhances Autophagy in Sepsis-Triggered Acute Kidney Injury

Sepsis-induced Acute kidney injury (SA-AKI), a common complication in sepsis, significantly impacts patients’ health and quality of life. M1 macrophages have been demonstrated to release inflammatory mediators that exacerbate kidney injury. MiR-155 has been implicated in promoting inflammation and damage during sepsis while reducing miR-155 levels alleviates SA-AKI. However, the relationship between miR-155 and M1 macrophage-derived exosomes in regulating autophagy during SA-AKI remains unclear. In this study, we aim to investigate the relationship between M1 macrophage-derived exosomes and miR-155 in regulating autophagy during SA-AKI. A mouse model of SA-AKI was established by performing cecal ligation and puncture (CLP) surgery. Additionally, the HK-2 cell line was utilized to establish a sepsis cell model by inducing lipopolysaccharide (LPS). We demonstrated that the mice model of SA-AKI exhibited renal injury along with enhanced autophagy, inflammation response, and macrophage polarization after CLP surgery. M1 macrophages attenuated cell viability and enhanced autophagy in LPS-treated HK-2 cells. Additionally, M1 macrophage-derived exosomes were observed to enhance autophagy in LPS-treated HK-2 cells. Furthermore, we confirmed an increased expression of miR-155 in M1 macrophage-derived exosomes. Furthermore, exosome-mediated miR-155 enhanced autophagy in LPS-treated HK-2 cells. In conclusion, this study provides the first evidence that exosomal miR-155 derived from M1 macrophages enhances autophagy in SA-AKI. These findings suggest that targeting exosomal miR-155 could be a promising therapeutic strategy for SA-AKI.

Macrophage-derived exosomal miR-155 regulating hepatocyte pyroptosis in MAFLD

BackgroundPrevious studies have shown that pyroptosis in hepatocyte is essential for the development of MAFLD. Growing evidence has shown that exosomal miRNAs-mediated communication between inflammatory cells and hepatocyte is an important link in MAFLD. In the present study, we aim to elucidate whether macrophage-derived exosomal miRNAs contribute to the hepatocyte pyroptosis in the pathophysiological process of MAFLD. MethodsThe effects of hepatocyte pyroptosis were investigated in an HFD-induced MAFLD mouse model and in the liver tissues from patients with MAFLD using immunohistochemistry, real-time PCR, Western blotting, and luciferase reporter assay, among other techniques. MiR-155 inhibitor tail injections and AAV-FoxO3a-GFP were also administered to respectively inhibit or overexpress its expression in an HFD-induced MAFLD mouse model. ResultsHepatocyte pyroptosis was heightened in the liver tissue of patients with MAFLD or HFD-induced MAFLD mouse. Importantly, treatment with a caspase-1 inhibitor or overexpression of FoxO3a reversed this trend. Our study also demonstrated that miR-155 expression and the number of infiltrated macrophages were increased, and knockdown of miR-155 attenuated hepotocyte pyroptosis and liver fibrosis in HFD-induced mouse. In addition, we demonstrated that macrophage-derived exosomal miR-155 was transferred to hepatocytes, leading to hepatocyte pyroptosis in MAFLD mouse. Furthermore, blockade of exosome secretion improved hepotocyte pyroptosis and liver fibrosis in HFD-induced mouse. On the contrary, macrophage-derived exosomal miR-155 worsened hepotocyte pyroptosis. Moreover, we found that miR-155 promoted hepatocyte pyroptosis in MAFLD by down-regulating FoxO3a. ConclusionsTaken together, our results demonstrated that macrophage-derived exosomal miR-155 promotes hepatocyte pyroptosis and liver fibrosis in MAFLD.

Macrophage-derived exosomes promote telomere fragility and senescence in tubular epithelial cells by delivering miR-155

BackgroundChronic kidney disease (CKD) is highly prevalent worldwide, and its global burden is substantial and growing. CKD displays a number of features of accelerated senescence. Tubular cell senescence is a common biological process that contributes to CKD progression. Tubulointerstitial inflammation is a driver of tubular cell senescence and a common characteristic of CKD. However, the mechanism by which the interstitial inflammation drives tubular cell senescence remains unclear. This paper aims to explore the role of exosomal miRNAs derived from macrophages in the development of tubular cell senescence.MethodsAmong the identified inflammation-related miRNAs, miR-155 is considered to be one of the most important miRNAs involved in the inflammatory response. Macrophages, the primary immune cells that mediate inflammatory processes, contain a high abundance of miR-155 in their released exosomes. We assessed the potential role of miR-155 in tubular cell senescence and renal fibrosis. We subjected miR-155−/− mice and wild-type controls, as well as tubular epithelial cells (TECs), to angiotensin II (AngII)-induced kidney injury. We assessed kidney function and injury using standard techniques. TECs were evaluated for cell senescence and telomere dysfunction in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization.ResultsCompared with normal controls, miR-155 was up-regulated in proximal renal tubule cells in CKD patients and mouse models of CKD. Moreover, the expression of miR-155 was positively correlated with the extent of renal fibrosis, eGFR decline and p16INK4A expression. The overexpression of miR-155 exacerbated tubular senescence, evidenced by increased detection of p16INK4A/p21expression and senescence-associated β-galactosidase activity. Notably, miR-155 knockout attenuates renal fibrosis and tubule cell senescence in vivo. Interestingly, once released, macrophages-derived exosomal miR-155 was internalized by TECs, leading to telomere shortening and dysfunction through targeting TRF1. A dual-luciferase reporter assay confirmed that TRF1 was the direct target of miR-155. Thus, our study clearly demonstrates that exosomal miR-155 may mediate communication between macrophages and TECs, subsequently inducing telomere dysfunction and senescence in TECs.ConclusionsOur work suggests a new mechanism by which macrophage exosomes are involved in the development of tubule senescence and renal fibrosis, in part by delivering miR-155 to target TRF1 to promote telomere dysfunction. Our study may provide novel strategies for the treatment of AngII-induced kidney injury.

The value of urinary exosomal microRNA-21 in the early diagnosis and prognosis of bladder cancer.

Bladder cancer (BC) poses high morbidity and mortality, with urinary exosomal microRNA (miR)-21 showing potential value in its diagnosis and prognosis, and we probed its specific role. We prospectively selected 116 BC patients and 116 healthy volunteers as the BC and control groups, respectively. BC urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 relative expression levels were assessed. The correlations between clinical indexes and urinary exosomal miR-21, prognostic value of miR-21, and diagnostic value of the five candidate miRNAs, urine cytology, and miRNA joint diagnostic panel for BC and urinary exosomal miR-21, miR-4454, and urine cytology for Ta-T1 and T2-T4 stage BC were analyzed. Urinary exosomal miR-146a-5p, miR-93-5p, miR-663b, miR-21, and miR-4454 were highly expressed in BC patients. miR-146a-5p, miR-93-5p, miR-663b, miR-21, miR-4454, miRNA combined diagnostic panel, and urine cytology had certain diagnostic value for BC, with miR-21, miR-4454, and miRNA co-diagnostic panel showing the highest diagnostic value. Collectively, urinary exosomal miR-21 was closely related to Tumor-Node-Metastasis staging and grading in BC patients. Urinary exosomal miR-21 had high diagnostic value for BC and Ta-T1 and T2-T4 stage BC, and had high predictive value for BC poor prognosis, providing an effective indicator for the occurrence, development, and prognostic assessment of BC.

Microglia-derived exosomes selective sorted by YB-1 alleviate nerve damage and cognitive outcome in Alzheimer’s disease

BackgroundNeuroinflammation is a characteristic pathological change of Alzheimer’s Diseases (AD). Microglia have been reported to participate in inflammatory responses within the central nervous system. However, the mechanism of microglia released exosome (EXO) contribute to communication within AD microenvironment remains obscure.MethodsThe interaction between microglia and AD was investigated in vitro and in vivo. RNA-binding protein immunoprecipitation (RIP) was used to investigate the mechanisms of miR-223 and YB-1. The association between microglia derived exosomal YB-1/miR-223 axis and nerve cell damage were assessed using Western blot, immunofluorescence, RT-PCR, ELISA and wound healing assay.ResultsHere, we reported AD model was responsible for the M1-like (pro-inflammatory) polarization of microglia which in turn induced nerve cell damage. While M2-like (anti-inflammatory) microglia could release miR-223-enriched EXO which reduced neuroinflammation and ameliorated nerve damage in AD model in vivo and in vitro. Moreover, YB-1 directly interacted with miR-223 both in cell and EXO, and participated in microglia exosomal miR-223 loading.ConclusionThese results indicate that anti-inflammatory microglia-mediated neuroprotection form inflammatory damage involves exporting miR-223 via EXO sorted by YB-1. Consequently, YB-1-mediated microglia exosomal sorting of miR-223 improved the nerve cell damage repair, representing a promising therapeutic target for AD.

Dumbbell Dual-Hairpin Triggered DNA Nanonet Assembly for Cascade-Amplified Sensing of Exosomal MicroRNA.

Exosomal microRNAs (miRNAs) are valuable biomarkers closely associated with cancer progression. Therefore, sensitive and specific exosomal miRNA biosensing has been employed for cancer diagnosis, prognosis, and prediction. In this study, a miRNA-based DNA nanonet assembly strategy is proposed, enabling the biosensing of exosomal miRNAs through dumbbell dual-hairpin under isothermal enzyme-free conditions. This strategy dexterously designs a specific dumbbell dual-hairpin that can selectively recognize exosomal miRNA, inducing conformational changes to cascade-generated X-shaped DNA structures, facilitating the extension of the X-shaped DNA in three-dimensional space, ultimately forming a DNA nanonet assembly. On the basis of the target miRNA, our design enriches the fluorescence signal through the cascade assembly of DNA nanonet and realizes the secondary signal amplification. Using exosomal miR-141 as the target, the resultant fluorescence sensing demonstrates an impressive detection limit of 57.6 pM and could identify miRNA sequences with single-base variants with high specificity. Through the analysis of plasma and urine samples, this method effectively distinguishes between benign prostatic hyperplasia, prostate cancer, and metastatic prostate cancer. Serving as a novel noninvasive and accurate screening and diagnostic tool for prostate cancer, this dumbbell dual-hairpin triggered DNA nanonet assembly strategy is promising for clinical applications.

Abstract LB297: Toll-like receptor 8 in tumor associated macrophages regulates miR-192-5p expression and drug resistance in neuroblastoma

Abstract MicroRNAs (miRs) are small non-coding RNAs (ncRNAs) with gene expression regulatory functions. MiRs are frequently dys-regulated in human cancers and orchestrate the biology of the disease, and its resistance to chemotherapy. Increasing evidence supports a role of the tumor microenvironment (TME) in promoting cancer growth and resistance to treatments. Tumor Associated Macrophages (TAMs) are a predominant cellular component of the TME of several types of cancers, including neuroblastoma (NB), the most frequent pediatric extra-cranial solid tumor. NB cells secrete miRs within small extracellular vesicles called exosomes, and these miR are shuttled to surrounding TAMs. Our lab was the first to show that cancer-derived exosomal miRs bind to Toll-like receptor 8 (TLR8) in TAMs, triggering an NF-κB-dependent production of TAM-derived miRs, that are then shuttled back to NB cells. Following up on that discovery, we identified a signature of miRs that are up/down-regulated in NB cells when co-cultured with TAMs expressing TLR8 or in which the TLR8 had been deleted with CRISPR/Cas9 engineering. We identified miR-192-5p as significantly down-regulated in NB cells when co-cultured with wild-type TAMs, however miR-192-5p expression was significantly increased in NB cells when co-cultured with TLR8-knock out TAMs, supporting a TLR8-mediated modulation of miR-192-5p in the NB TME. Additionally, we found that over-expression of miR-192-5p in NB cells increased sensitivity of NB cells to temozolomide and cisplatin, two commonly used drugs in the treatment of this malignancy. Overall, these findings support the use of TLR8 inhibitors to up-regulate miR-192-5p and increase sensitivity of NB cells to chemotherapy. Citation Format: Nicolo Toldo, Verena Martinez Rodriguez, Muller Fabbri. Toll-like receptor 8 in tumor associated macrophages regulates miR-192-5p expression and drug resistance in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB297.

Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. 6 horses with SEA and 6 healthy controls. The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

Abstract 459: Exosomal miRNA 16-5p/29a-3p secreted from pancreatic cancer induce adipose atrophy by inhibiting adipogenesis and promoting lipolysisExosomal miRNA 16-5p/29a-3p secreted from pancreatic cancer induce adipose atrophy by inhibiting adipogenesis and promoting lipolysis

Abstract Over 80% of the patients with pancreatic ductal adenocarcinoma (PDAC) have cachexia, which is associated with reduced survival, decreased quality of life, and higher rates of metastatic disease. To date, the diagnostic and therapeutic options of PDAC-associated cachexia remain limited due to the lack of a complete understanding of the underlying pathological mechanism. Here, we demonstrate that fat loss is the earliest feature of cachexia induced by PDAC through its secreted exosomes. By analyzing exosomal components of normal and cancer-derived exosomes through small RNA-seq, we identify miR-16-5p, miR-21-5p, miR-29a-3p and miR-125b-5p enriched in pancreatic cancer-derived exosomes and in the exosomes from sera of mice or patients with pancreatic tumor. Among these, miR-16-5p and miR-29a-3p play a key role in fat loss by inhibiting adipogenesis through decreasing Erlin2 and CMPK1 expression leading to C/EBPβ and PPARγ downregulation. Synergistically, miR-29a-3p increases ATGL expression to facilitate lipolysis by suppressing MCT1 expression. Furthermore, PDAC-derived exosomes deprived of miR-16-5p and miR-29a-3p fail to induce fat loss, suggesting that these two exosomal miRs are required for PDAC-induced fat loss. These findings unravel a precise mechanism of how PDAC causes adipose atrophy via exosomal miRs and may provide new diagnostic and therapeutic strategies for PDAC-induced cachexia. Citation Format: Sui-Chih Tien, Chin-Chun Chang, I-Chih Chen, Ching-Hsuan Huang, Hsuan-Yu Peng, Yu-Ting Chang, Ming-Chu Chang, Chun-Mei Hu, Wen-Hwa Lee. Exosomal miRNA 16-5p/29a-3p secreted from pancreatic cancer induce adipose atrophy by inhibiting adipogenesis and promoting lipolysisExosomal miRNA 16-5p/29a-3p secreted from pancreatic cancer induce adipose atrophy by inhibiting adipogenesis and promoting lipolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 459.

Islet-resident macrophage-derived miR-155 promotes β cell decompensation via targeting PDX1

Chronic inflammation is critical for the initiation and progression of type 2 diabetes mellitus via causing both insulin resistance and pancreatic β cell dysfunction. miR-155, highly expressed in macrophages, is a master regulator of chronic inflammation. Here we show that blocking a macrophage-derived exosomal miR-155 (MDE-miR-155) mitigates the insulin resistances and glucose intolerances in high-fat-diet (HFD) feeding and type-2 diabetic db/db mice. Lentivirus-based miR-155 sponge decreases the level of miR-155 in the pancreas and improves glucose-stimulated insulin secretion (GSIS) ability of β cells, thus leading to improvements of insulin sensitivities in the liver and adipose tissues. Mechanistically, miR-155 increases its expression in HFD and db/db islets and is released as exosomes by islet-resident macrophages under metabolic stressed conditions. MDE-miR-155 enters β cells and causes defects in GSIS function and insulin biosynthesis via the miR-155-PDX1 axis. Our findings offer a treatment strategy for inflammation-associated diabetes via targeting miR-155.

Diagnostic value of plasma-derived exosomal miR-223 for epithelial ovarian cancer

ObjectivesTo evaluate the diagnostic value of plasma exosomal miR-223 and its combination with CA125 for the diagnosis of early-stage epithelial ovarian cancer (EOC).Patients and methodsExosomes derived from the plasma of 78 EOC patients, 40 patients with epithelial benign ovarian tumors, and 52 healthy participants were isolated using the ultracentrifugation method and identified by transmission electron microscopy (TEM) and western blot.ResultsThe expression of exosomal miR-223 was significantly upregulated in the plasma of EOC patients compared to that in healthy subjects and patients with benign diseases. The combination of exosomal miR-223 and CA125 from plasma had an equivalent area under the ROC curve (AUC) to CA125 alone for discriminating between EOC and non-EOC cases, including healthy subjects and benign ovarian tumors. However, the AUC value of the combination was 0.944 (95% CI: 0.899–0.990) for differentially diagnosing early-stage EOC from healthy subjects, slightly higher than that of CA125 alone (0.928, 95% CI: 0.875–0.981), with a sensitivity and specificity of 0.9784 and 0.885, respectively.ConclusionOur data suggest that plasma exosomal miR-223 can be used as a complement to CA125 to increase the diagnostic power for differentiating early-stage EOC from healthy subjects.