Exosomal miR-21 Research Articles

Abstract 1700: Excessive ROS in ovarian cancer cells impairs anti-tumor capacities of immune cells through exosomal miR-155/PD-L1 pathway

Abstract Background and Objective: Genetic and metabolic alterations in cancer cells promote the sustained oxidative stress as reflected by excessive production of reactive oxygen species (ROS). We and other groups have identified that the ROS-sensitive microRNAs regulate the cancer cell response to ROS. However, it has yet to be determined how these miRNAs convey the ROS signal to downstream effectors and thus produce biological consequences. Accumulative evidence indicates that tumor-derived exosomes are emerging mediators of tumorigenesis. In this study, we aimed to investigate how ROS convey the oncogenic signals through cellular interactions via exosomes within the tumor microenvironment. Methods and Results: Through NanoString nCounter miRNA analysis, we identified miR-155-5p as the most abundant miRNAs enriched in the exosomes derived from epithelial ovarian cancer (EOC) cells treated with a ROS scavenger N-acetylcysteine (NAC). Mechanistically, tumor exosomal miR-155-5p was transferred to tumor-associated macrophages (TAM), directly targeting PD-L1 to inhibit the infiltration of TAM into tumor microenvironment. The role of tumor-derived exosomal miR-155-5p was determined by co-culture experiments and gain or loss of function studies in vitro, and confirmed in xenograft mouse models in nude mice and immunocompetent mice. In addition, we established a 3D-3-culture system to entail the co-culture of NAC-treated EOC cell spheroids, TAM and T cells. We found that CD8+ T-cell ratio was increased and production of IFN-γ, IL-2, and TNF-α was promoted. What's more, the immunocompetent EOC mouse model was established and treated with NAC-modified mouse ovarian cancer cell-released exosomes. The results showed that NAC-modified tumor growth was significantly reduced compared to control group. Further analysis showed that miR-155-5p increased in TAM, while PD-L1 expression levels decreased in macrophages from tumors and spleens. Moreover, the T cell function was also augmented as evaluated by the expression of Ki-67 and Granzyme B on CD8+ T cells. Conclusion: In conclusion, our study reveals a novel mechanism underlying PD-L1 expression in EOC. Ovarian cancer cells with excessive ROS levels release exosomes that mediate the interaction between cancer cells and immune cells; it is this interplay that produces an immunosuppressive tumor microenvironment by upregulation of PD-L1 in macrophages, and eventually contributes to the progress of EOC through the ROS/miR-155-5p/PD-L1 pathway. Citation Format: Xiang Li, Gao-chan Wang, Jun He. Excessive ROS in ovarian cancer cells impairs anti-tumor capacities of immune cells through exosomal miR-155/PD-L1 pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1700.

Paeonol inhibits NLRP3 mediated inflammation in rat endothelial cells by elevating hyperlipidemic rats plasma exosomal miRNA-223

Atherosclerosis (AS) is a multifactorial chronic inflammatory disease, and hyperlipidemia is the important factors leading to AS, which can cause vascular endothelial dysfunction. Paeonol (Pae) is a potential therapeutic drug for AS, and we have previously shown that Pae regulated the expression of monocytes-derived exosomal microRNA-223 (miR-223). However, the mechanisms of the anti-AS effect of Pae are still not fully understood. In this study, we aim to investigate if Pae could inhibit NLRP3 inflammasome mediated inflammation via elevating hyperlipidemic rats plasma-derived exosomal miR-223. We used high-fat-diet induced hyperlipidemic rats as model for further investigation. Rats were treated with Pae (75, 150 or 300 mg/kg) orally, and then exosomes were isolated from hyperlipidemic rat plasma by ultracentrifugation. In vivo experiments confirmed that Pae markedly reduced serum TC, TG, IL-1β, and IL-6 levels. Both CCK-8 and trypan blue staining showed that the survival rate of rat aortic endothelial cells (RAECs) in the Pae-exo group was higher than that in the model group. Also, Pae-exo dose-dependently increased the survival rate of RAECs and reduced inflammatory cytokines level (IL-1β, and IL-6). Furthermore, Pae-exo successfully increased the expression of exosomal miR-223 and relieved inflammatory secretion. Finally, decreased expression of NLRP3, ASC, caspase-1 and ICAM-1 indicated that Pae-exo attenuated inflammatory reaction of RAECs by suppressing NLRP3 signaling pathway. Altogether, our results showed that Pae inhibited the downstream NLRP3 inflammasome pathway by increasing the level of miR-223 in plasma derived exosomes of hyperlipidemic rats, providing new insights in the treatment of AS with the use of Pae.

Macrophage-derived exosomes attenuate fibrosis in airway epithelial cells through delivery of antifibrotic miR-142-3p

IntroductionIdiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology and cure. Recent studies have reported a dysregulation of exosomal microRNAs (miRs) in the IPF context....

Serum Exosomal miR-1290 is a Potential Biomarker for Lung Adenocarcinoma.

PurposeLung cancer is a leading cause of cancer-related death, with lung adenocarcinoma (LUAD) representing the most common subtype. Recently, exosome-based biomarkers have provided new diagnostic approaches for malignancies. We aimed to identify specific exosomal microRNAs (miRNAs) as noninvasive biomarkers for LUAD.Patients and MethodsA total of 110 participants were enrolled and randomly divided into two sets: the discovery set (n=20) and the validation set (n=90). Exosomes were isolated from serum, and miRNAs were subsequently extracted. Candidate miRNAs (miR-21, miR-221-3p, miR-222-3p, miR-223, miR-638 and miR-1290) were detected by quantitative real-time PCR (qRT-PCR) in the discovery set. The upregulated miR-1290 was then selected for further analysis in the validation set along with three tumor markers (CEA, CYFRA21-1 and NSE). The diagnostic and prognostic value of exosomal miR-1290 were estimated through receiver-operating characteristic (ROC) and survival analysis.ResultsSerum exosomal miR-1290 was significantly upregulated in LUAD patients compared to healthy controls (P<0.001) and decreased after resection (P=0.0029). Its expression level was associated with tumor stage, tumor size, lymph node and distant metastasis (all P <0.05). Exosomal miR-1290 had a higher diagnostic efficacy than CEA, CYFRA21-1 and NSE, with a sensitivity of 80.0% and specificity of 96.7% (AUC: 0.937, 95% CI: 0.890–0.985; P<0.001). Moreover, LUAD patients with a high level of exosomal miR-1290 had significantly poorer progression-free survival (PFS) than those with a low level of exosomal miR-1290 (mean PFS: 14 months vs 37 months, P<0.001). Cox proportional hazards model analysis demonstrated that exosomal miR-1290 could be an independent risk factor for the prognosis of LUAD (HR=7.80, P=0.017).ConclusionSerum exosomal miR-1290 could be a potential diagnostic and prognostic biomarker for LUAD.

Abstract 475: Beta-adrenoceptor Stimulation Uniquely Regulates Exosome Micro-rna Content Secreted From Cardiomyocytes in vitro and Those Found in vivo Circulating in Blood

Background and Purpose: The influence of β-adrenoceptor (βAR) signaling on the regulation of exosomes secreted from cardiomyocytes is unknown and since catecholamines are increased in heart failure (HF), there is interest in uncovering whether βARs can induce specific changes in the content of circulating blood exosomes in HF. In this study, we have evaluated whether βAR stimulation by isoproterenol (ISO) on neonatal rat ventricle myocytes (NRVMs) and in vivo in mice can alter the number, size and microRNA (miR) content of secreted exosomes. Methods and Results: ISO treatment of NRVMs did not change exosome number and size of compared than vehicle (PBS). After purifying total RNA from treated myocytes and secreted exosomes, we evaluated the expression level of 37 candidate miR’s, which were selected from previous microarray data. We found that ISO treatment decreased 14 miR’s (miR-222, -106a, -292a, -181b, -210, -489, -214, -1947, -195, -17, -7b, -93, -532 and -19a) in exosomes and in the myocytes themselves, mir-292a and -1947 were up regulated and mir-7b, down regulated. Further, ISO was then used to treat C57 mice via osmotic pump chronically. The number and size of exosomes purified from circulating blood was not changed after 2 and 8 weeks. We evaluated expression of the 14 miRs down-regulated in myocytes as well miR-1 and -21 (important cardiac miRs) both in the blood and hearts of ISO-treated mice. MiR-1 did not change in both blood exosomes and heart tissue and miR-21 was up-regulated in heart tissue but not in blood both at 2 and 8 weeks after ISO treatment. In addition, miRNA-489 and -7b was down-regulated in hearts with miR-214 and -292a up regulated. In blood exosomes, we found only down-regulation of miR-489 and -7b but not miR-214 and 292a. Conclusions: We found that the βAR agonist ISO altered exosomal miR contents but not exosome size and number from myocytes. Importantly, this was found in myocytes in culture and in vivo blood and myocardium that were treated with ISO but several differences were found and changes in blood and myocytes were not homogeneous. However, two miRs, mir-7b and -miR-489, both changes similarly from their origin (NRVMs or mouse hearts) and also in circulating blood exosomes. Therefore, these miRs may represent biomarkers for sympathetic nervous system abnormalities in HF and their therapeutic potential should be evaluated.

MicroRNA-21-Enriched Exosomes as Epigenetic Regulators in Melanomagenesis and Melanoma Progression: The Impact of Western Lifestyle Factors.

DNA mutation-induced activation of RAS-BRAF-MEK-ERK signaling associated with intermittent or chronic ultraviolet (UV) irradiation cannot exclusively explain the excessive increase of malignant melanoma (MM) incidence since the 1950s. Malignant conversion of a melanocyte to an MM cell and metastatic MM is associated with a steady increase in microRNA-21 (miR-21). At the epigenetic level, miR-21 inhibits key tumor suppressors of the RAS-BRAF signaling pathway enhancing proliferation and MM progression. Increased MM cell levels of miR-21 either result from endogenous upregulation of melanocytic miR-21 expression or by uptake of miR-21-enriched exogenous exosomes. Based on epidemiological data and translational evidence, this review provides deeper insights into environmentally and metabolically induced exosomal miR-21 trafficking beyond UV-irradiation in melanomagenesis and MM progression. Sources of miR-21-enriched exosomes include UV-irradiated keratinocytes, adipocyte-derived exosomes in obesity, airway epithelium-derived exosomes generated by smoking and pollution, diet-related exosomes and inflammation-induced exosomes, which may synergistically increase the exosomal miR-21 burden of the melanocyte, the transformed MM cell and its tumor environment. Several therapeutic agents that suppress MM cell growth and proliferation attenuate miR-21 expression. These include miR-21 antagonists, metformin, kinase inhibitors, beta-blockers, vitamin D, and plant-derived bioactive compounds, which may represent new options for the prevention and treatment of MM.

Plasma Small Extracellular Vesicles Derived miR-21-5p and miR-92a-3p as Potential Biomarkers for Hepatocellular Carcinoma Screening.

IntroductionLiquid biopsy using circulating microvesicles and exosomes is emerging as a new diagnostic tool that could improve hepatocellular carcinoma (HCC) early diagnosis and screening protocols. Our study aimed to investigate the utility of plasma exosomal miR-21-5p and miR-92-3p for HCC diagnosis during screening protocols.MethodsThe study group included 106 subjects: 48 patients diagnosed with HCC during screening, who underwent a potentially curative treatment (surgical resection or liver transplantation), 38 patients with liver cirrhosis (LC) on the waiting list for liver transplantation, and 20 healthy volunteers. The exosomes were isolated by precipitation with a reagent based on polyethylene glycol and were characterized based on morphological aspects (i.e., diameter); molecular weight; CD63, CD9, and CD81 protein markers; and exosomal miR-21-5p and miR-92a-3p expression levels.ResultsWe first demonstrate that the exosome population isolated with the commercially available Total Exosome Isolation kit respects the same size ranging, morphological, and protein expression aspects compared to the traditional ultracentrifugation technique. The analysis of the expression profile indicates that miR-21-5p was upregulated (p = 0.017), and miR-92a-3p was downregulated (p = 0.0005) in plasma-derived exosomes from HCC subjects, independently from the patient’s characteristics. AUROC for HCC diagnosis based on AFP (alpha-fetoprotein) was 0.72. By integrating AFP and the relative expression of exosomal miR-21-5p and miR-92a-3p in a logistic regression equation for HCC diagnosis, the combined AUROC of the new exosomal miR HCC score was 0.85—significantly better than serum AFP alone (p = 0.0007).ConclusionTogether with serum AFP, plasma exosomal miR-21-5p and miR-92a-3p could be used as potential biomarkers for HCC diagnosis in patients with LC subjected to screening and surveillance.

Bu-Shen-Yi-Sui Capsule, an Herbal Medicine Formula, Promotes Remyelination by Modulating the Molecular Signals via Exosomes in Mice with Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a common inflammatory demyelinating disorder of the central nervous system. Bu-shen-yi-sui capsule (BSYSC) could significantly reduce the relapse rate, prevent the progression of MS, and enhance remyelination following neurological injury in experimental autoimmune encephalomyelitis (EAE), an established model of MS; however, the mechanism underlying the effect of BSYSC on remyelination has not been well elucidated. This study showed that exosomes carrying biological information are involved in the pathological process of MS and that modified exosomes can promote remyelination by modulating related proteins and microRNAs (miRs). Here, the mechanism by which BSYSC promoted remyelination via exosome-mediated molecular signals was investigated in EAE mice and oligodendrocyte progenitor cells (OPCs) in vitro. The results showed that BSYSC treatment significantly improved the body weight and clinical scores of EAE mice, alleviated inflammatory infiltration and nerve fiber injury, protected the ultrastructural integrity of the myelin sheath, and significantly increased the expression of myelin basic protein (MBP) in EAE mice. In an in vitro OPC study, BSYSC-containing serum, especially 20% BSYSC, promoted the proliferation and migration of OPCs and induced OPCs to differentiate into mature oligodendrocytes that expressed MBP. Furthermore, BSYSC treatment regulated the expression of neuropilin- (NRP-) 1 and GTX, downregulated the expression of miR-16, let-7, miR-15, miR-98, miR-486, and miR-182, and upregulated the level of miR-146 in serum exosomes of EAE mice. In conclusion, these results suggested that BSYSC has a neuroprotective effect and facilitates remyelination and that the mechanism underlying the effect of BSYSC on remyelination probably involves regulation of the NRP-1 and GTX proteins and miRs in serum exosomes, which drive promyelination.

Human umbilical cord mesenchymal stem cells-derived exosomes deliver microRNA-375 to downregulate ENAH and thus retard esophageal squamous cell carcinoma progression

BackgroundExosomal microRNAs (miRNAs or miRs) from bone marrow-derived mesenchymal stem cells (UCMSCs) have emerged as promising therapeutic strategies for cancer treatment. The current study aimed to elucidate the underlying mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs)-derived exosomal miR-375 in esophageal squamous cell carcinoma (ESCC).MethodsAfter determining the expression of miR-375 and its putative target enabled homolog (ENAH) in ESCC tissues and cells, we tested effects of their altered expression on ESCC proliferation, invasion, migration, and tumorsphere formation was subsequently measured. Transfected hUCMSCs-derived exosomes (hUCMSCs-exo) were isolated and co-cultured with ESCC cells to measure the effects of miR-375 delivered by hUCMSCs-exo on ESCC development. Finally, we investigated the effect of miR-375 on tumor growth in vivo.ResultsThe expression of miR-375 was reduced, while the expression of ENAH was elevated in ESCC. ENAH was identified as a target gene of miR-375. Elevated miR-375 or depleted ENAH expression inhibited ESCC cell proliferation, invasion, migration, tumorsphere formation, and promoted apoptosis. Moreover, miR-375 delivered by hUCMSCs-exo could suppress ESCC cell proliferation, invasion, migration, tumorsphere formation, but promoted apoptosis in vitro, as well as inhibiting tumor growth in vivo.ConclusionsTaken together, hUCMSCs-exo can deliver miR-375 to suppress ENAH expression and subsequently inhibit the initiation and progression of ESCC.

Therapeutic angiogenesis with exosomal microRNAs: an effectual approach for the treatment of myocardial ischemia.

Therapeutic angiogenesis presents a potential approach for treating ischemic heart diseases especially in patients who are not appropriate candidates for traditional approaches of revascularization. This approach acts through inducing the neovascularization or maturation of pre-existing collateral vessels into functional arteries to bypass the blocked arteries and restore perfusion to ischemic myocardium. Successful stimulation of local angiogenesis can be established by the cross talk between stem cells, endothelial cells, and cardiomyocytes, which is mainly mediated by paracrine communication accompanied by secreted exosomes. Exosomes are extracellular vesicles carrying a complex of signaling molecules, such as microRNAs (miRs) that can modulate the function of recipient cells. Such particles have been indicated to exert cardioprotective role through providing signaling cues for angiogenesis, an effect ascribed mainly to their miRs content. Exosomal miRs-mediated therapeutic angiogenesis has been under drastic preclinical and clinical studies. In the current review, it was aimed to summarize pro-angiogenic exosomal miRs released by various cell types mediating angiogenesis, including stem cells, endothelial cells, and cardiomyocytes, which appear to exert a therapeutic effect on the myocardial ischemia. In brief, secreted exosomal miRs including miR-210, miR-23a-3p, miR-424, let-7f, miR-30b, miR-30c, miR-126, miR-21, miR-132, miR-130a-3p, miR-214, miR-378, miR-126, miR-133, and let-7b-5p could protect against myocardial ischemia through inducing cardiac angiogenesis and vascular regeneration resulting in the increase blood flow to ischemic myocardium.

&lt;p&gt;Exosomal miR-638 Inhibits Hepatocellular Carcinoma Progression by Targeting SP1&lt;/p&gt;

PurposeExosomal microRNAs (miRNAs) play essential roles in the development of hepatocellular carcinoma (HCC). Nevertheless, the role and mechanism of exosomal miR-638 in HCC development remain largely unknown.MethodsExosomes were isolated and confirmed via transmission electron microscopy and western blot. The abundances of miR-638 and specificity protein 1 (SP1) were measured via quantitative reverse transcription polymerase chain reaction or western blot. Cell proliferation was investigated by Cell Counting Kit-8, colony formation assay, apoptosis, cell cycle distribution and related protein expression. Cell migration and invasion were detected via transwell assay and western blot. Co-culture experiment was performed to assess exosome transfer from HCC cells to endothelial cells. The target correlation between miR-638 and SP1 was analyzed via dual-luciferase reporter and RNA immunoprecipitation assays. The subcutaneous xenograft experiment was conducted to test the function of miR-638 in vivo.ResultsThe miR-638 level declined in exosomes from serum or HCC cell medium. miR-638 overexpression repressed HCC cell proliferation by decreasing viability and colony formation and inducing apoptosis and cell cycle arrest at G1 phase, and decreased abilities of migration and invasion. Exosomal miR-638 from HCC cells could transfer to human umbilical vein endothelial cells (HUVECs) and suppress HUVEC proliferation, migration and invasion. SP1 was a target of miR-638 and overexpression of SP1 reversed the effect of miR-638 on HCC cells. Overexpression of miR-638 reduced xenograft tumor growth via decreasing SP1.ConclusionExosomal miR-638 inhibited HCC tumorigenesis by targeting SP1. This study indicated the potential clinical implications of miR-638 in HCC.

Neural progenitor cells-secreted exosomal miR-210 induced by hypoxia influences cell viability.

Hypoxia as a microenvironment is essential for the development of neural stem/progenitor cells (NSCs/NPCs). Our previous studies showed that mild hypoxia can promote proliferation of NPCs. However, the underlying mechanisms are remaining unknown. In the present study, we explored the impact of hypoxia on miR-210 secretion and its effect on cell viability. We found that short time or long time of hypoxia treatment could increase the expression of miR-210, but also promoted its secretion into the medium. The results of exosomes isolation and quantitative real-time PCR showed that hypoxia increased the levels of miR-210 in the exosome enriched from the medium. In addition, the secreted miR-210 can be absorbed by recipient NPCs. The resutls of cell viability assay showed that low levels of secreted miR-210 slightly increased cell viability of NPCs. In contrast, high levels of secreted miR-210 exhibited an inhibitory effect on cell viability. These effects were blocked by an miR-210-specific inhibitor. Taken together, hypoxia increased secretion of miR-210 in exosomes and exhibited a differential effect on cell viability of recipient NPCs.Video abstract: http:/links.lww.com/WNR/A588.

Ovatodiolide, isolated from Anisomeles indica, suppresses bladder carcinogenesis through suppression of mTOR/β-catenin/CDK6 and exosomal miR-21 derived from M2 tumor-associated macrophages

Bladder cancer (BCa) is the fourth leading cause of cancer deaths worldwide due to its aggressiveness and resistance against therapies. Intricate interactions between cancer cells and the tumor microenvironment (TME) are essential for both disease progression and regression. Thus, interrupting molecular communications within the TME could potentially provide improved therapeutic efficacies. M2-polarized tumor-associated macrophages (M2 TAMs) were shown to contribute to BCa progression and drug resistance. We attempted to provide evidence for ovatodiolide (OV) as a potential therapeutic agent that targets both TME and BCa cells. First, tumor-suppressing functions of OV were determined by cell viability, colony, and tumor-sphere formation assays using a coculture system composed of M2 TAMs/BCa cells. Subsequently, we demonstrated that extracellular vesicles (EVs) isolated from M2 TAMs containing oncomiR-21 and mRNAs, including Akt, STAT3, mTOR, and β-catenin, promoted cisplatin (CDDP) resistance, migration, and tumor-sphere generation in BCa cells, through increasing CDK6, mTOR, STAT3, and β-catenin expression. OV treatment also prevented M2 polarization and reduced EV cargos from M2 TAMs. Finally, in vivo data demonstrated that OV treatment overcame CDDP resistance. OV only and the OV + CDDP combination both resulted in significant reductions in mTOR, β-catenin, CDK6, and miR-21 expression in tumor samples and EVs isolated from serum. Collectively, we demonstrated that M2 TAMs induced malignant properties in BCa cells, in part via oncogenic EVs. OV treatment prevented M2 TAM polarization, reduced EV cargos derived from M2 TAMs, and suppressed β-catenin/mTOR/CDK6 signaling. These findings provide preclinical evidence for OV as a single or adjuvant agent for treating drug-resistant BCa.

Exosomal miR-21/Let-7a ratio distinguishes non-small cell lung cancer from benign pulmonary diseases.

AimTo assess the exosomal miR‐21/Let‐7a ratio, a noninvasive method, in distinguishing non‐small cell lung cancer (NSCLC) from benign pulmonary diseases.MethodsThe exosomes were extracted from the peripheral blood serum using serum exosomal extraction kit. miR‐21 and Let‐7a levels were evaluated by quantitative reverse transcription polymerase chain reaction.ResultsWe found that miR‐21/Let‐7a ratio of NSCLC patients was significantly higher than that of healthy people, patients with pulmonary inflammation diseases, and benign pulmonary nodules, respectively. Receiver‐operating characteristic analysis revealed that as compared with healthy controls, miR‐21/Let‐7a produced the area under the curve (AUC) at 0.8029 in patients with NSCLC, which helped to distinguish NSCLC from healthy controls with 81.33% sensitivity and 69.57% specificity. In addition, the AUC of miR‐21/Let‐7a in NSCLC patients was 0.8196 in comparison to patients with pulmonary inflammation diseases. Meanwhile, the sensitivity and specificity were 56.00% and 100%, respectively. Furthermore, compared with patients with benign pulmonary nodules, the AUC of miR‐21/Let‐7a in NSCLC patients was 0.7539. The sensitivity and specificity were 56.00% and 82.61%, respectively.ConclusionIn the present study, our findings revealed that exosomal miR‐21/Let‐7a ratio holds considerable promise as a noninvasive biomarker for the diagnosis of NSCLC from benign pulmonary diseases.

Serum Exosomal MicroRNA-21, MicroRNA-126, and PTEN Are Novel Biomarkers for Diagnosis of Acute Coronary Syndrome.

Acute coronary syndrome (ACS) is a serious threat to public health. Based on clinical manifestations, ACS can be classified into unstable angina (UA) pectoris and acute myocardial infarction (AMI). The purpose of this study was to explore the possibility of using serum exosomal microRNA (miR)-126, miR-21, and phosphatase and tensin homolog (PTEN) expression levels as biomarkers of UA and AMI and to investigate whether these levels were positively correlated with the severity of coronary stenosis based on the Gensini score. Exosomes were isolated by ultracentrifugation from the serum of 34 patients with AMI, 31 patients with UA, and 22 healthy controls. The isolated exosomes were characterized by electron microscopy and particle size analysis; exosomal identity was further confirmed by western blotting using exosome-specific antibodies. Real-time quantitative polymerase chain reaction indicated that the serum exosomal levels of miR-126 and miR-21 were significantly higher in the patients with UA and AMI than in the healthy controls. Enzyme-linked immunosorbent assay showed that the serum exosomal PTEN levels were significantly higher in the UA and AMI groups than in the control group. Receiving operating characteristic curve analysis demonstrated the diagnostic efficiency of serum exosomal miR-126, miR-21, and PTEN levels for predicting AMI and UA. In addition, the circulating exosomal miR-126 level was positively correlated with the severity of coronary artery stenosis in patients with UA and AMI based on the Gensini score.

Effect of exosome-derived miR-223 from macrophages on the metastasis of gastric cancer cells

Objective: To investigate the effect and mechanism of exosome-derived miR-223 from macrophage on gastric cancer (GC) cell metastasis. Methods: Exosomes isolated from macrophages culture medium were characterized and cocultured with GC cell, the miRNA level was detected by qRT-PCR. The migration and invasion of GC cell were detected by transwell. The internalization of exosomes, transfer of miR-223 was observed by immunofluorescence. Macrophage were transfected with a miR-223 inhibitor or negative control, transwell and scratch test were employed to explore the effect of macrophage derived exosome on the migration and invasion of GC cell. Western blot and RT-PCR assay were performed to uncover the underlying mechanisms of miR-223 and PTEN-PI3K/AKT pathway. Results: This study showed that macrophage and macrophage-derived exosomes promoted the migration and invasion of gastric cancer cell(253.2±6.3, 451.8±12.8, 453.4±14.4, all P<0.01, and 98.4±5.1, 276.5±10.3, 257.3±8.5, all P<0.01, respectively). miR-223 was enriched in macrophage-derived exosomes, which was transferred to the co-cultivated gastric cancer cells. miR-223 knockdown in macrophage reversed the migration and invasion of exosomes on gastric cancer cells(215.6±9.2, 402.5±11.6, 253.7±10.4, all P<0.01, and 91.5±8.2,263.4±9.3,105.8±9.3,all P<0.01, respectively).Functional studies revealed that exosomal miR-223 derived from macrophage promoted the metastasis of GC cells via the PTEN-PI3K/AKT pathway. In addition, itshowed thatthe actin cytoskeleton was altered, and multiple proteins associated with epithelial-mesenchymaltransition (EMT) were upregulated. Conclusion: Exosomal transfer of macrophage-derived miR-223 promote the metastasis of GC cells through targeting the PTEN-PI3K/AKT pathway.

Abstract B045: Exosomal microRNAs are associated with prostate cancer aggressiveness in African American patients

Abstract Background: The morbidity and mortality rates of prostate cancer (PCa) in African American (AA) are 2-3 times higher than European American (EuA) men. The molecular mechanisms underlying the aggressiveness of PCa have not fully identified. Thus, our aim was to evaluate the diagnostic/prognostic utility of exosomal microRNAs (miRs) to classify PCa patients according their race and aggressive phenotype in AA patients. Their functional role in tumor aggressiveness was also determined. Methods: Exosomes were isolated from the conditioned media of AA and EuA PCa cell lines. The expression of miRs was validated in exosomes, free-circulating plasma, and FFPE tissue specimens of forty AA and EuA patients using quantitative real-time PCR analysis. The sensitivity and specificity of exosomal miRs to classify prostate cancer patients according their race and aggressiveness were assessed using receiver operating characteristic (ROC) curve analysis. To study the functional significance of exosomal miRs, cell proliferation, clonogenic, cell cycle and migration assays were performed in PCa cells transfected with miR-3128. Results: Differential expression of exosomal miR-3613-3p, miR-3218, miR-3679, and miR-3680 was demonstrated in the plasma of AA versus EuA of PCa patients. While exosomal miR-3613 and miR-3679 (p&amp;lt;0.05) were upregulated, free-circulating miRs downregulated (p&amp;lt;0.05) in the plasma of AA versus EuA patients. The accuracy of miR-3679 to discriminate AA from EuA was improved when combined with the other three miRs (AUC jumped from 0.717 to 0.897). Intriguingly, miR-3128 showed a dual role in AA versus EuA cells. Overexpression of miR-3128 increased the cell growth in AA cells while it did the opposite in EuA cells. These data were recapitulated by migration, cell cycle and clonogenic assays. Conclusion: Our findings underline the role of exosomal miRs in health disparity of PCa. The differential expression of miRs in AA men demonstrates their reliability as biomarkers and their potential role in promoting tumor aggressiveness in AA men. Citation Format: Hamdy E.A. Ali, Rofaida Gaballa, Andrew S. Sholl, Mohamed Gaballah, Juan J. Bustamante, Preeti Zanwar, Hamed I. Ali, Zakaria Y. Abd Elmageed. Exosomal microRNAs are associated with prostate cancer aggressiveness in African American patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B045.

P1605THE EARLY RISE OF URINARY EXOSOMAL BK VIRUS MICRORNA AS A PREDICTIVE MARKER FOR BK VIRUS NEPHROPATHY IN A PROSPECTIVE KIDNEY TRANSPLANTATION COHORT

Abstract Background and Aims BK virus nephropathy (BKVN) is one of the significant contributors to allograft dysfunction in kidney transplantation. Because of the lack of effective anti-viral medication, early detection of BKV replication and preemptive management is vital to preserving allograft function. Herein, we investigated the predictive value of urinary exosomal BKV-microRNA for BKVN in the kidney transplantation cohort. Method ARTKT (assessment of immunologic risk and tolerance in kidney transplantation) is a prospective observational cohort in which a total of 420 kidney transplant recipients were enrolled from 7 teaching hospitals in South Korea since 2015. Urine samples were collected at 0.5, 3, 6, 12, and 24 months after kidney transplantation in this cohort. All enrolled patients were reviewed to identify the patients diagnosed with BKVN histologically (biopsy-proven BKVN) and those experienced BK viremia defined as plasma BKV DNA load &amp;gt; 4 log10 copies per mL (presumptive BKVN). Urinary exosomal miR was quantified using real-time reverse transcription PCR. Results 10 patients and 14 patients developed biopsy-proven BKVN and presumptive BKVN, respectively. Because of unavailable urine samples, urinary exosomal BKV-microRNA was investigated in 8 patients with biopsy-proven BKVN and 13 patients with presumptive BKVN. 61 patients with no evidence of BKVN were selected as a negative control. Most of BKVN was developed in a median time of 3.4 months after transplantation. In line with this pattern, urinary exosomal BKV-microRNA peaked at 3 months after transplantation and decreased thereafter in biopsy-proven BKVN and presumptive BKVN, keeping higher value in biopsy-proven BKVN compared to presumptive BKVN after 3 months. At 2 weeks after transplantation, half of the patients with biopsy-proven BKVN showed an early rise of urinary exosomal BKV-microRNA, whereas patients with presumptive BKVN and no evidence of BKVN did not. However, plasma BK virus DNA was not detected in all groups at 2 weeks after transplantation. In multivariate cox proportional hazards model, urinary exosomal BKV-microRNA was significantly associated with biopsy-proven BKVN. Conclusion A urinary exosomal BKV-microRNA increase as early as 2 weeks after kidney transplantation predicts future BKVN development, enabling early intervention.

Abstract C029: Exosomes-associated miR-5001, miR-3692 and miR-4529 are novel biomarkers for aggressive prostate cancer and associated with poor prognosis in African American patients

Abstract Background: Although microRNA (miR) profiling has been widely used to predict clinical outcomes, differential miR expressions that can segregate prostate cancer (PCa) patients based on their races and tumor aggressiveness have not been fully investigated. We aimed to determine the diagnostic and prognostic abilities of exosomal miRs to identify the aggressive phenotypes of PCa in African American (AA) men. Methods: Exosomes were isolated from blood of twenty AA and European Americans (EuA) PCa patients at low and high Gleason scores and their aged-matched healthy subjects (n=20) as well as AA and EuA normal and PCa cells. miR profiling was performed on PCa exosomes derived from blood and PCa cells. The expression level was correlated with clinical outcomes of PCa patients. The sensitivity and specificity of exosomal miRs were assessed using receiver operating characteristic (ROC) curve. Results: Results from miR profiling showed a number of exosomal miRs that were able to differentiate normal from PCa, low from high Gleason scores and AA from EuA PCa patients. These dysregulated miRs were validated in another cohort of forty PCa patients in addition to a large panel of PCa cell lines. In the validation cohort, miR-5001, miR-3692 and miR-4529 were upregulated in the exosomes derived from blood of AA compared to EuA men. These miRs were correlated with age, T-stage, residual tumor, involvement of lymph nodes, Gleason score, and overall survival of AA patients. The combination of these miRs showed high discriminatory power (AUC=0.91) for segregation of PCa patients according to their clinical outcomes. Conclusion: miR profiling identified a new set of miRs that can differentiate PCa specimens based on their race and Gleason score. The differential expression of these miRs demonstrates their potential role as biomarkers in the context of racial disparity. Further studies are warranted to determine their role in PCa at advanced stages. Citation Format: Rofaida Gaballa, Mohamed Gaballah, Hamdy E.A. Ali, Andrew S. Sholl, Hamed I. Ali, Zakaria Y. Abd Elmageed. Exosomes-associated miR-5001, miR-3692 and miR-4529 are novel biomarkers for aggressive prostate cancer and associated with poor prognosis in African American patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C029.

MiR-210 in exosomes derived from CAFs promotes non-small cell lung cancer migration and invasion through PTEN/PI3K/AKT pathway

ObjectiveCancer-associated fibroblasts (CAFs) function as a crucial factor in tumor progression by carrying exosomes to neighboring cells. This study was assigned to expound the underlying mechanism of CAFs-derived exosomal miR-210 in non-small cell lung cancer (NSCLC) progression. MethodCAFs and normal fibroblasts (NFs) were isolated and identified. Exosomes secreted from CAFs and NFs were isolated to analyze their effects on tumor volume and epithelial-mesenchymal transition (EMT). Exosomal miR-210 expression level was measured. The effects of exosomal miR-210 and UPF1 on cell viability, EMT, PTEN/PI3K/AKT signal pathway were determined. Dual-luciferase reporter gene assay was utilized to validate the binding of UPF1 to miR-210. ResultsCAFs-derived exosomes (CAFs-exo) were successfully extracted and proven to be uptake by lung cancer cells. Up-regulated expression level of miR-210 was found in CAFs-exo, which was then proved to enhance cell migration, proliferation, invasion abilities and EMT in NSCLC cells. Overexpression of miR-210 can also inhibit UPF1 and PTEN, but activate the PTEN/PI3K/AKT pathway. UPF1 was a target gene of miR-210. MiR-210 can up-regulate UPF1 expression level to activate PTEN/PI3K/AKT pathway. ConclusionMiR-210 secreted by CAFs-exo could promote EMT by targeting UPF1 and activating PTEN/PI3K/AKT pathway, thereby promoting NSCLC migration and invasion.