Exosomal miRNA Expression Profile Research Articles

Expression profiling of exosomal miRNAs derived from different stages of infection in mice infected with Echinococcus granulosus protoscoleces using high-throughput sequencing.

Echinococcosis is a worldwide zoonosis. The mechanism of the establishment, growth, and persistence of parasites in the host has not been fully understood. Exosomes are found to be a way of information exchange between parasites and hosts. They exist in various body fluids widely. There are few studies on host-derived exosomes and their miRNA expression profiles at different infection time points. In this study, BALB/c mice were intraperitoneally infected with protricercariae. Exosomes were extracted from plasma (0, 3, 9, and 20weeks post infection), and the expression profiles of exosome miRNA in the peripheral blood of mice were determined using RNA-sequencing. Compared to the 0week groups, 24, 35, and 22 differentially expressed miRNAs were detected in infected mouse at the three infection stages, respectively. The results showed that there were significant differences in the miRNAs of exosomes at different infection time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to annotate the different miRNAs. The results showed that the biological pathways of parasites changed significantly at different stages of infection, with many significant and abundant pathways involved in cell differentiation, inflammation, and immune response, such as MAPK signaling pathway, Th17 cell differentiation, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway, etc. These results suggest that miRNA may be an important regulator of interactions between Echinococcus granulosus and host. The data provided here provide valuable information to increase understanding of the regulatory function of microRNAs in the host microenvironment and the mechanism of host-parasite interaction. This may help us to find targets for Echinococcus granulosus to escape host immune attack and control Echinococcus granulosus infection in the future.

Expression Profiles of Exosomal MicroRNAs Derived from Cerebrospinal Fluid in Patients with Congenital Hydrocephalus Determined by MicroRNA Sequencing.

Purpose Congenital hydrocephalus is one of the most common birth defects worldwide. Exosomal microRNAs (miRNAs) in body fluids have been implicated in many diseases. However, their involvement in cerebrospinal fluid from congenital hydrocephalus is not well understood. This study is aimed at investigating the role of dysregulated exosomal miRNAs in congenital hydrocephalus. Methods We collected cerebrospinal fluid samples from 15 congenital hydrocephalus patients and 21 control subjects. We used miRNA sequencing to generate exosomal miRNA expression profiles in three pairs of samples. We identified 31 differentially expressed exosomal miRNAs in congenital hydrocephalus and predicted their target mRNAs. Results Three microRNAs (hsa-miR-130b-3p, hsa-miR-501-5p, and hsa-miR-2113) were selected according to their fold changes and the function of their target mRNAs, and only hsa-miR-130b-3p and hsa-miR-501-5p were confirmed their expression levels in all samples. Moreover, upregulated hsa-miR-130b-3p might mediate the downregulation of the phosphatase and tensin homolog gene (PTEN), which has been associated with hydrocephalus, via binding to its 3′-untranslated region by dual-luciferase reporter assay. Conclusion This study implicates that abnormally expressed exosomal miRNAs in cerebrospinal fluid may be involved in the pathomechanism of congenital hydrocephalus.

Exosomal miRNA Expression Profiling and the Roles of Exosomal miR-4741, miR-32, miR-3149, and miR-6727 on Gastric Cancer Progression.

Objective Accumulated evidence highlights the biological implications of exosomes in gastric cancer. Herein, we conducted the exosomal miRNA expression profiling and identified potential diagnostic markers for gastric cancer. Methods Plasma exosomes were isolated and identified from three gastric cancer patients and three healthy participants. Microarrays of exosomal miRNAs were then analyzed. Differentially expressed exosomal miRNAs were screened with fold − change|≥2.0 and p ≤ 0.05. Among them, miR-4741, miR-32, miR-3149, and miR-6727 expressions were verified in tissues and plasma of patients and healthy subjects. ROC curves were conducted for evaluating the diagnostic performance. The roles of miR-32, miR-3149, miR-6727, and miR-4741 on gastric cancer progression were observed by cellular experiments. Results Isolated exosomes were well characterized by Western blot and transmission electron microscopy as well as nanoparticle-tracking analyses. According to the microarrays, 142 exosomal miRNAs were upregulated, and 34 were downregulated in gastric cancer than healthy subjects. miR-4741 upregulation and miR-32, miR-3149, and miR-6727 downregulations were found in tissues and plasma of gastric cancer patients. The AUCs of miR-4741, miR-32, miR-3149, and miR-6727 were separately 0.8554, 0.9456, 0.7683, and 0.8923. Upregulated miR-32, miR-3149, and miR-6727 as well as downregulated miR-4741 lowered proliferative, migratory, and invasive capacities as well as elevated apoptotic levels of gastric cancer cells. Conclusion Our study successfully isolated and verified exosomes from plasma of gastric cancer as well as proposed four exosomal miRNAs that could act as promising diagnostic markers and suppress gastric cancer progression.

Identification and bioinformatics analysis of differentially expressed milk exosomal microRNAs in milk exosomes of heat-stressed Holstein cows.

In summer, heat stress is one of the primary reasons for the compromised health and low milk productivity of dairy cows. Hyperthermia affects milk synthesis and secretion in the mammary glands of dairy cows. As molecules for intercellular communication, milk-derived exosomes carry genetic material, proteins, and lipids, playing a crucial role in mammary tissue growth and milk synthesis in dairy cows. The aim of this study was to explore the milk exosomal miRNA profile of heat-stressed and normal Holstein cows. We isolated and identified milk exosomes to screening for differentially expressed miRNAs using small RNA sequencing. Then, TargetScan and miRanda algorithms were used to predict the putative targets of the differentially expressed miRNAs, whereas GO and KEGG pathway enrichment analyses were performed for the differentially expressed miRNA-target genes. Our results showed that 215 miRNAs were significantly differentially expressed in heat-stressed milk exosomes, of which one was upregulated and 214 were significantly downregulated. GO and KEGG enrichment analyses indicated that differentially expressed miRNAs might play a role in apoptosis, autophagy, and the p38 MAPK pathway. qRT-PCR assay verified that the expression of miRNAs was consistent with the sequencing results, warranting further verification of their specific targets of action. In conclusion, changes in the miRNA expression profile of milk exosomes indicated the role of exosomal miRNAs in regulating heat stress resistance and apoptosis in dairy cows. Our results suggested that milk-derived exosomal miRNAs could increase mammary gland resistance to heat stress, thereby enhancing milk synthesis in dairy cows.

Exploring the pathogenesis of type 2 diabetes mellitus intestinal damp-heat syndrome and the therapeutic effect of Gegen Qinlian Decoction from the perspective of exosomal miRNA

Ethnopharmacological relevanceDiabetes is a common, complex, chronic metabolic disease. A randomized, double-blind, placebo-parallel controlled clinical study has shown that Gegen Qinlian Decoction (GQD) can reduce glycosylated hemoglobin in type 2 diabetes mellitus (T2DM) intestinal damp-heat syndrome patients in a dose-dependent manner. AimTo explore the pathogenesis of T2DM intestinal damp-heat syndrome and the therapeutic effect of GQD from the perspective of exosomal microRNA (miRNA). MethodsEligible patients were selected and treated with GQD for 3 months to evaluate their clinical efficacy. Effective cases were matched with healthy volunteers, and saliva samples were collected. Exosomal miRNA was extracted from saliva and analyzed by chip sequencing. Subsequently, the function of the differential gene and the signal transduction pathway were analyzed using bioinformatics technology. Finally, three target miRNAs were randomly selected from the T2DM group/healthy group, and two target miRNAs in the T2DM before treatment/after treatment group were randomly selected for qPCR verification. Finally, we conducted a correlation analysis of the miRNAs and clinical indicators. The registration number for this research is ChiCTR-IOR-15006626. Results(1) The expression of exosomal miRNA chips showed that there were 14 differentially expressed miRNAs in the T2DM group/healthy group, and 26 differentially expressed miRNAs in the T2DM before treatment/after treatment group. (2) Enrichment results showed that in the T2DM group/healthy group, it was primarily related to cell development, body metabolism, TGF-β, and ErbB signaling pathways. In the T2DM before treatment/after treatment group, it was mainly related to cellular metabolic regulation processes, and insulin, Wnt, and AMPK signaling pathways. (3) The qPCR verification showed that the expressions of hsa-miR-9-5p, hsa-miR-150–5p, and hsa-miR-216b-5p in the T2DM group was higher (P<0.05). Following GQD treatment, hsa-miR-342–3p and hsa-miR-221–3p were significantly downregulated (P<0.05). (4) hsa-miR-9-5p was positively correlated with BMI (P<0.05), and hsa-miR-150–5p was positively correlated with total cholesterol and triglycerides (P<0.05). The GQD efficacy-related gene hsa-miR-342–3p was positively correlated with the patient's initial blood glucose level (P<0.05), and hsa-miR-221–3p was positively correlated with total cholesterol and triglycerides (P<0.05). ConclusionThe exosomal miRNA expression profile and signaling pathways related to T2DM intestinal damp-heat syndrome and the efficacy of GQD were established, which provides an alternative strategy for precision traditional Chinese medicine treatment.

MicroRNA sequence analysis of plasma exosomes in early Legg–Calvé–Perthes disease

The pathogenesis of Legg–Calvé–Perthes disease (LCPD) has not been fully elucidated, and studies on epigenetic changes that may contribute to the pathogenesis of LCPD are rare. MicroRNAs (miRNAs) are epigenetic modifications that play a critical role in gene regulation. This study aimed to determine the expression profiles of circulating exosomal miRNAs and examine the role of exosomal miRNAs in LCPD. Exosomes were extracted from the plasma of three patients with LCPD and three matched healthy volunteers. Total exosomal miRNAs were isolated, and next-generation sequencing and bioinformatic approaches were performed. The top 10 most differentially upregulated miRNAs were identified, and qRT-PCR validation was performed using additional 10 matches. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, plasma exosomes were used in verifying osteoclastogenesis and the endothelial dysfunction phenotypes involved. The elevated miRNAs in LCPD plasma exosomes were tested for osteoclastogenesis and endothelial dysfunction in vitro. Sequencing results revealed the expression profiles of plasma exosomal miRNAs with differential expression from the DESeq-identified miRNA profiles in LCPD versus controls in a pairwise comparison. Gene Ontology and KEGG pathway analyses indicated that the predicted target genes of different miRNAs were mainly enriched in the endothelial and osteoclast cells related to signaling pathways. Functional phenotype experiments showed that the plasma exosomes in the LCPD group promoted osteoclastogenesis and endothelial cell dysfunction. qRT-PCR experiments showed that nine miRNAs in circulating exosomes in LCPD patients were higher than those in the healthy controls. miR-3133, miR-4644, miR-4693-3p, and miR-4693-5p promoted endothelial dysfunction, and miR-3133, miR-4693-3p, miR-4693-5p, miR-141–3p and miR-30a promoted osteoclastogenesis in vitro. This study demonstrated that plasma exosomes from LCPD promote endothelial cell dysfunction and osteoclastogenesis likely through their miRNAs, which might contribute to the development of LCPD.

A Pilot Study of miRNA Expression Profile as a Liquid Biopsy for Full-Marathon Participants.

Exosomal microRNA (miRNA) in plasma and urine has attracted attention as a novel diagnostic tool for pathological conditions. However, the mechanisms of miRNA dynamics in the exercise physiology field are not well understood in terms of monitoring sports performance. This pilot study aimed to reveal the miRNA dynamics in urine and plasma of full-marathon participants. Plasma and urine samples were collected from 26 marathon participants before, immediately after, 2 h after, and one day after a full marathon. The samples were pooled, and exosomal miRNAs were extracted and analyzed using next-generation sequencing. We determined that the exosomal miRNA expression profile changed under time dependency in full marathon. New uncharacterized exosomal miRNAs such as hsa-miR-582-3p and hsa-miR-199a-3p could be potential biomarkers reflecting physical stress of full marathon in plasma and urine. In addition, some muscle miRNAs in plasma and urine have supported the utility for monitoring physical stress. Furthermore, some inflammation-related exosomal miRNAs were useful only in plasma. These results suggest that these exosomal miRNAs in plasma and/or urine are highly sensitive biomarkers for physical stress in full marathons. Thus, our findings may yield valuable insights into exercise physiology.

Serum exosomal miRNA from endometriosis patients correlates with disease severity

PurposeExosomes are vesicles secreted by cells that contain a wide variety of biomolecules, including proteins or nucleic acids. MicroRNAs (miRNAs), which are commonly found in exosomes, are known to play important roles in the pathophysiology of endometriosis.MethodsThis study investigated the miRNA expression profile of serum exosomes from women with endometriosis in comparison with normal controls as well as the possible role of identified miRNAs in the pathogenesis of endometriosis. Exosomes with a diameter between 60 and 100 nm were identified by their expression of exosomal marker proteins CD9 and CD63.ResultsMicroarray miRNA expression profiling analysis revealed that 26 genes were significantly up-regulated and 19 genes were significantly down-regulated in serum exosomes from endometriosis patients compared with normal controls. These differentially expressed miRNAs were mainly enriched in the regulation of cellular development, metabolism, and involved in the regulation of the MAPK and PI3k-Akt pathways. qRT-PCR analysis verified the differential expression of three miRNAs, miR-26b-5p, miR-215-5p, and miR-6795-3p.ConclusionFurther analysis indicated that these differentially expressed miRNAs in serum exosomes may be involved in the pathogenesis of endometriosis and are related to the severity and certain symptoms of endometriosis.

Urinary exosome microRNA signatures as a noninvasive prognostic biomarker for prostate cancer

Predicting the risk of metastasis before starting prostate cancer (PCa) treatment can minimize the overtreatment of indolent cases and help choosing appropriate treatment. The levels of circulating microRNAs (miRNAs) from body fluids can be used as noninvasive prognostic biomarkers. In this study, urinary exosomal miRNA expression profiles of 149 PCas were determined and the miRNAs associated with metastasis were identified: miR-21, miR-16, miR-142-3p, miR-451, and miR-636. When evaluating clinical factors together, miR-21, miR-451, miR-636, and preoperative prostate-specific antigen (PSA) level remained significant in the multivariate analysis. Based on them, we developed a “Prostate Cancer Metastasis Risk Scoring (PCa-MRS)” model. The PCa-MRS showed superior stratification power (AUC = 0.925) to preoperative PSA or clinical Gleason score. Patients with high scores showed significantly poorer biochemical recurrence-free survival than those with low scores (P = 6.53 × 10−10). Our results showed the potential of urinary exosomal miRNAs as noninvasive markers for predicting metastasis and prognosis in PCa patients.

Identification of Urinary Exosomal miRNAs for the Non-Invasive Diagnosis of Prostate Cancer.

BackgroundNovel and non-invasive biomarkers with higher sensitivity and specificity for the diagnosis of prostate cancer (PCa) is urgently needed. In this study, we used next-generation sequencing (NGS) to characterize the genome-wide exosomal miRNA expression profiling in urine specimens and explored the diagnostic potential of urinary exosomal miRNAs for PCa.MethodsUrinary exosomal microRNA expression profiling was performed by next-generation sequencing (NGS) and then validated by quantitative real-time PCR.ResultsSignificant downregulation of urinary exosomal miR-375 was observed in PCa patients compared with healthy controls, while the expression levels of urinary exosomal miR-451a, miR-486-3p and miR-486-5p were found to be significantly up-regulated in the PCa patients. Furthermore, the expression level of urinary exosomal miR-375 showed a significant correlation with the clinical T-stage and bone metastasis of patients with PCa (P<0.05). Receiver operator characteristic curve demonstrated that the urinary exosomal miR-375, miR-451a, miR-486-3p and miR-486-5p levels can be used to differentiate PCa patients from healthy controls, with area under the curves (AUCs) of 0.788, 0.757, 0.704 and 0.796, respectively. The urinary exosomal miR-375 was found to be superior in discriminating between localized and metastatic PCa with an AUC of 0.806. Moreover, PCa patients can be distinguished from patients with benign prostatic hyperplasia by using a panel combining urinary exosomal miR-375 and miR-451a with an AUC of 0.726.ConclusionThese findings demonstrate that the urinary exosomal miRNAs can serve as novel and non-invasive biomarkers for diagnosing and predicting the progression of PCa.

Differential Expression of Urinary Exosomal Small RNAs in Idiopathic Membranous Nephropathy.

Background Idiopathic membranous nephropathy (IMN) is a major cause of adult nephrotic syndromes, and reliable noninvasive biomarkers for diagnosis and monitoring are urgently needed. In this study, we performed small RNA (sRNA) sequencing to explore sRNA profiles of urinary exosomes derived from IMN patients and healthy controls (CON) to provide clues for identifying novel noninvasive sRNA biomarkers for IMN. Methods Urine samples were collected from five healthy controls and six patients with IMN. High-throughput sequencing was used to screen sRNA expression profiles of urinary exosomes from patients with IMN in two independent cohorts. Results Urinary exosomes were successfully isolated and used to obtain exosomal sRNAs. We screened 131 differentially expressed miRNAs, including 28 specifically expressed miRNAs, then explored the top 10 specifically expressed miRNAs in all IMN individuals. The specifically expressed miRNAs and differentially expressed miRNAs provide potential biomarkers for IMN. Additionally, we discovered numerous sRNAs derived from genomic repetitive sequences, which could represent an exciting new area of research. Conclusion Herein, we revealed significant differences in expression profiles of urinary exosomal miRNAs and repetitive region-derived sRNAs between patients with IMN and healthy controls. The findings could facilitate the development of potential molecular targets for membranous nephropathy.

Exosomal MicroRNA-374b-5p From Tubular Epithelial Cells Promoted M1 Macrophages Activation and Worsened Renal Ischemia/Reperfusion Injury.

Tubular epithelial cells (TECs) represent the primary site of renal ischemia/reperfusion injury (RIRI). However, whether the damage of TECs could drive the initiation of inflammation was unclear. Here we investigated the role of the TECs and macrophages during RIRI. Increased expression of inflammation response and activated M1 macrophage were determined in the mice model of RIRI. Moreover, we demonstrated global miRNA expression profiling of renal exosomes, and miR-374b-5p was most upregulated in these exosomes in vivo. Inhibition of miR-374b-5p in the mice upon RIR operation would alleviate the kidney injury via decreasing the production of proinflammatory cytokines and suppressing the macrophage activation. Similar results were also identified in the hypoxia-induced cell model where exosomal miR-374b-5p was dramatically upregulated. Uptake of exosomes derived from the hypoxic TECs by macrophages would trigger M1 polarization via transferring miR-374b-5p. Besides, we confirmed that miR-374b-5p could directly bind to Socs1 using a dual-luciferase reporter assay. Notably, when we injected the miR-374b-5p-enriched exosomes into mice, a high-level inflammatory response and M1 macrophage activation were performed. Our studies demonstrated that exosomal miR-374b-5p played an essential role in the communication between injured TECs and macrophages, resulting in the M1 macrophage activation during RIRI. The blockage of the release of such exosomes may serve as a new therapeutic strategy for RIRI.

Serum exosome microRNA panel as a noninvasive biomarker for molecular diagnosis of fulminant myocarditis

Exosome-derived microRNAs (miRNAs) are potential diagnostic biomarkers. However, little is known about their effectiveness as diagnostic biomarkers of fulminant myocarditis (FM). This study aimed to explore serum exosomal miRNAs as potential biomarkers for FM diagnosis. Peripheral blood samples were collected from 99 patients with FM, 32 patients with nonfulminant myocarditis (NFM), and 105 healthy controls (HCs). The miRNA expression profiles of serum exosomes were determined using next-generation sequencing, and differentially expressed miRNAs were further analyzed by quantitative reverse transcriptase polymerase chain reaction. A logistic regression model was constructed using a training cohort (n = 120) and then validated using an independent cohort (n = 106). The area under the receiver operating characteristic curve was used to evaluate diagnostic accuracy. In FM patients, hsa-miR-30a, hsa-miR-192, hsa-miR-146a, hsa-miR-155, and hsa-miR-320a were validated as significantly and differentially expressed candidates that could serve as potential markers for diagnosing FM. In addition, the miRNA panel (hsa-miR-155 and hsa-miR-320a) from the multivariate logistic regression model demonstrated high accuracy in the diagnosis of FM and was able to distinguish FM from HCs and NFM. Moreover, the diagnostic value of the miRNA panel was greater than that of CRP and cTn alone or together. The miRNA panel provided the excellent diagnostic capability for FM.

Human Periodontal Ligament Stem Cell-Derived Exosomes Promote Bone Regeneration by Altering MicroRNA Profiles.

The role and underlying mechanism of exosomes derived from human periodontal ligament stem cells (PDLSC) in osteogenesis are unclear. In the present study, we identified the exosomes derived from PDLSCs and found that osteogenic induction can enhance the osteogenic ability of PDLSC-derived exosomes in promoting the osteogenic differentiation of rat bone marrow stem cells (BMSCs). To investigate the underlying mechanism, we analyzed the exosomal miRNA expression profiles of undifferentiated and osteogenic differentiated PDLSCs by RNA sequencing. The results showed that seventy-two miRNAs were upregulated and thirty-five miRNAs were downregulated after osteogenic induction. The results of Gene Ontology analysis and pathway analysis demonstrated that the target genes of differentially expressed exosomal miRNAs participate in the regulation of a variety of biological processes, such as catalytic activity, protein binding, metabolic processes, cell development, and differentiation, and are enriched in osteogenic differentiation-related pathways, such as MAPK signaling, AMPK signaling, and insulin signaling pathways. Our results reveal for the first time that the exosomal miRNAs derived from osteogenic differentiated PDLSCs may promote the osteogenic differentiation of BMSCs, which provides a basis for further research on the regulatory function of exosomal miRNA of PDLSCs during osteogenesis.

Abstract 15839: Patients With Abdominal Aortic Aneurysms Have Reduced Levels of MicroRNA 122-5p in Circulating Exosomes

Introduction: There are currently no specific biomarkers to screen and monitor disease progression in abdominal aortic aneurysms (AAA), which are the 15 th leading cause of death in the United States. Circulating exosomes contain microRNAs (miRNA) that are potential biomarkers of disease. This study aimed to characterize the exosomal miRNA expression profile of patients with AAA in order to identify novel markers of disease. Methods: A total of 109 patients scheduled to undergo a duplex ultrasound (US) for screening or surveillance of AAA were screened to participate in the study. Eleven patients with AAA (max aortic diameter &gt;3cm) and 15 non-aneurysmal controls (max aortic diameter &lt;3cm on screening US) were enrolled. Circulating plasma exosomes were isolated using Cushioned-Density Gradient Ultracentrifugation and total RNA was extracted. Next Generation Sequencing was performed on the Illumina HiSeq4000 SE50 after NEBNext Multiplex Small RNA Library Prep. Differential miRNA expression analysis was performed using DESeq2 software with a Benjamini-Hochberg correction. MicroRNA expression profiles were validated by Quantitative Real-Time PCR. Results: Aortic diameter, as measured by US, was significantly larger in the AAA group (mean maximum diameter 5.2 vs 2.3 cm, p =2.84x10 -6 ). Aneurysm patients were more likely to suffer from CAD (5/11 vs 1/15, p = 0.05) but had similar levels of PAD (4/11 vs 2/15, p =0.35) and COPD (4/11 vs 4/15, p =0.68) compared to controls. A total of 40 miRNAs were differentially expressed ( p &lt;0.05). Of these, 18 miRNAs were up-regulated and 22 were down-regulated in controls compared to AAA. After false discovery rate (FDR) adjustment, only miR-122-5p was expressed at significantly different levels in AAA compared to controls (fold change = 5.03 controls vs AAA; raw p = 1.8x10 -5 ; FDR p =0.02). Conclusions: Plasma exosomes from AAA patients have significantly reduced levels of miRNA-122-5p compared to controls.

Expression profile of serum-related exosomal miRNAs from parathyroid tumor.

The expression pattern of exosomal miRNAs derived from parathyroid tumor is still unknown. In the present work, we aimed to examine the differences on microRNA (miRNA) expression, present in serum exosomes, by comparing parathyroid carcinoma (PC) and parathyroid adenoma (PA). MiRNA expression profile of serum exosomes, derived from 4 PC patients and 4 PA patients, were analyzed by next-generation sequencing technology. The differential expressions of target miRNAs were further verified in both serum exosomes and tissues of PC/PA patients by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Lastly, receiver operating characteristic (ROC) curves were plotted to investigate the efficiency of target exosomal miRNAs in distinguishing PC patients from PA controls. Multiple differentially expressed miRNAs of serum exosomes were screened out by sequencing. Based on this screening, hsa-miR-146b-5p (p = 0.0846), hsa-miR-27a-5p (p = 0.0412), hsa-miR-93-5p (p = 0.73), hsa-miR-381-3p (p = 0.1239) and hsa-miR-134-5p (p = 0.0694) were upregulated in the serum exosomes of PC patients. These results were validated by qPCR, where the trend on differential miRNA expression was consistent with the sequencing results. Specifically, the expression of exosomal hsa-miR-27a-5p was able to clearly distinguish PC patients from PA controls, and related analysis indicated that the area under the ROC curve was 0.8594 (p = 0.0157). Here we present, for the first time, the miRNA expression profile of serum exosomes derived from PC patients. Based on this result, we presently suggest that the exosomal hsa-miR-27a-5p may serve as a putative tumor marker for preoperative identification of PC and PA subjects.

High-Throughput Sequencing-Based Identification of Serum Exosomal Differential miRNAs in High-Grade Glioma and Intracranial Lymphoma.

Objective At present, no effective noninvasive method is currently available for the differential diagnosis of high-grade glioma and intracranial lymphoma. In the present study, we aimed to screen microRNA (miRNA) markers in serum exosomes for differential diagnosis of high-grade glioma and intracranial lymphoma using high-throughput sequencing technology. Methods Patients with intracranial lymphoma or high-grade glioma and healthy controls were included in this study (training cohort (n = 10) and validation cohort: intracranial lymphoma (n = 10), high-grade glioma (n = 32), and healthy controls (n = 20)). After RNA was extracted from serum exosomes, the high-throughput sequencing was used to determine the expression profiles of serum exosomal miRNAs and screen the differentially expressed miRNAs. RT-qPCR was used to verify the expressions of the selected miRNAs. The differences of miRNA expressions between groups were assessed by the Kruskal-Wallis test. The diagnostic value was analyzed using the receiver operating characteristic (ROC) curve. Results High-throughput sequencing demonstrated that 170 miRNAs, including 109 upregulated ones and 61 downregulated ones, were differentially expressed in serum exosomes between the patients with intracranial lymphoma and high-grade glioma. Compared with the healthy controls, the number of differential serum exosomal miRNAs in the high-grade glioma group and intracranial lymphoma group was 130 and 173, respectively. RT-qPCR proved that both miR-766-5p and miR-376b-5p were significantly downregulated in high-grade glioma and intracranial lymphoma patients compared with the healthy controls (all p < 0.001), and the expression of serum exosomal miR-766-5p in the intracranial lymphoma group was lower compared with the high-grade glioma group (p < 0.05). The areas under ROC curve (AUCs) of serum exosomal miR-766-5p and miR-376b-5p for the diagnosis of glioma were 0.8883 (p < 0.001) and 0.7688 (p = 0.001), respectively, and they were 0.9271 (p < 0.001) and 0.8542 (p < 0.001), respectively, for the diagnosis of intracranial lymphoma. Moreover, the AUC value of serum exosomal miR-766-5p for the differential diagnosis of glioma and intracranial lymphoma was 0.7201 (p = 0.026). Conclusions miR-766-5p and miR-376b-5p in serum exosomes might be used as auxiliary diagnostic indicators for high-grade glioma and intracranial lymphoma, and miR-766-5p might be used as a differential diagnostic marker for both diseases.

MicroRNA Derived from Circulating Exosomes as Noninvasive Biomarkers for Diagnosing Renal Cell Carcinoma.

PurposeRenal cell carcinoma (RCC) is the most common type of kidney cancer in adults. Exosomes are membrane-enclosed extracellular vesicles, and exosomal RNA can be a biomarker for cancer diagnosis and prognosis in RCC patients. We aim to identify differences in miRNA expression profiles in peripheral blood exosomes between RCC patients and healthy subjects as well as to investigate novel markers of RCC.MethodsWe performed exosomal miRNA sequencing of plasma samples obtained from five RCC patients and five control subjects, subsequently 22 RCC patients and 16 control subjects were investigated using qPCR to confirm the differential miRNA which from plasma exosomal RNA sequencing. ROC curves were constructed to assess the diagnostic accuracy of exosomal miRNAs as diagnostic biomarkers of RCC.ResultsExosomes were isolated with the exoeasy maxi kit and confirmed using TEM and NTA. They have a spherical structure with a diameter of approximately 40–180 nm. The exosomal miRNA sequence results showed that a total of 2357 miRNAs were detected, and 245 miRNAs were differentially expressed between RCC patients and healthy controls (p<0.001, average counts >5, log|fc|>1). Further analysis revealing that, versus the control, 17 miRNAs are up-regulated and 5 miRNAs are down-regulated under selection conditions with average miRNAs counts >100. qPCR was performed using 38 subjects—the results showed that the expression levels of hsa-mir-149-3p and hsa-mir-424-3p were upregulated; the expression levels of hsa-mir-92a-1-5p were significantly downregulated in the plasma exosomes of RCC. For diagnosis of RCC, the AUC of hsa-mir-92a-1-5p, hsa-mir-149-3p and hsa-mir-424-3p was 0.8324, 0.7188 and 0.7727, with the sensitivity of 0.875, 0.750 and 0.750, and the specificity of 0.773,0.727 and 0.818, respectively, at the best cutoff value.ConclusionOur study revealed that the expression levels of hsa-mir-92a-1-5p, hsa-mir-149-3p and hsa-mir-424-3p were significantly abnormal in RCC patients, which may be novel biomarkers for RCC diagnosis.

Plasma Exosomal miRNA Expression Profile as Oxaliplatin-Based Chemoresistant Biomarkers in Colorectal Adenocarcinoma.

Background: Chemotherapy is one of the most common therapies used in the treatment of colorectal cancer (CRC), but chemoresistance inevitably occurs. It is challenging to obtain an immediate and accurate diagnosis of chemoresistance. The potential of circulating exosomal miRNAs as oxaliplatin-based chemoresistant biomarkers in CRC patients was investigated in this study.Methods: Plasma exosomal miRNAs in sensitive and resistant patients were analyzed by miRNA microarray analysis, followed by verification with a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay in two independent cohorts. The diagnostic accuracy was determined by ROC curve analysis. Logistic regression analysis and Spearman's rank correlation test were also performed. Finally, bioinformatics was used to preliminarily explore the potential molecular mechanism of the selected miRNAs in chemoresistance.Results: miRNA microarray analysis identified four upregulated miRNAs and 20 downregulated miRNAs in chemoresistant patients compared to chemosensitive patients. Twelve markedly dysregulated miRNAs were selected for further investigation, of which six (miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i) were verified to be significantly and consistently dysregulated (>1.5-fold, P < 0.05). The combination of the six miRNAs had the highest AUC (0.825, 95% CI, 0.753–0.897). The expression level of these 6 miRNAs was not correlated with tumor location, stage, or chemotherapy program. Only miR-100 was significantly upregulated in low histological grade. GO analysis and KEGG pathway analysis showed that miRNAs were related to RNA polymerase II transcription and enriched in the PI3K-AKT signaling pathway, AMPK signaling pathway, and FoxO signaling pathway.Conclusions: We identified a panel of plasma exosomal miRNAs, containing miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i, that could significantly distinguish chemoresistant patients from chemosensitive patients. The detection of circulating exosomal miRNAs may serve as an effective way to monitor CRC patient responses to chemotherapy. Targeting these miRNAs may also be a promising strategy for CRC treatment.

Downregulation of exosomal miR‑1273a increases cisplatin resistance of non‑small cell lung cancer by upregulating the expression of syndecan binding protein

Resistance to platinum-based drugs, such as cisplatin (CDDP), has been one of the major factors adversely affecting the clinical prognosis of patients with advanced non-small cell lung cancer (NSCLC). While it has been demonstrated that dysregulation of microRNAs (miRNAs) may contribute to cisplatin resistance in NSCLC, the underlying mechanisms remain largely unclear. In the present study, the effect of exosomal miR-1273a on cisplatin sensitivity of NSCLC was investigated. Microarray analysis was conducted to analyze the miRNA expression profiles in exosomes isolated from A549 cells treated with or without CDDP, and miR-1273a was found to be the most prominently downregulated miRNA in CDDP-treated exosomes. Overexpression of miR-1273a significantly increased the cytotoxicity of CDDP and induced apoptosis in A549 cells. Syndecan binding protein (SDCBP) was predicted to be a direct target of miR-1273a by bioinformatics and was found to be downregulated by miR-1273a in A549 cells. Furthermore, decreased plasma exosomal miR-1273a and increased plasma SDCBP levels were found to be associated with worse therapeutic outcomes of patients with advanced NSCLC receiving platinum-based chemotherapy. These findings suggest that miR-1273a is closely associated with the development of cisplatin resistance and may serve as a potential prognostic biomarker and therapeutic target for NSCLC.