Discovery and validation of extracellular vesicle-associated miRNAs as noninvasive detection biomarkers for early-stage non-small-cell lung cancer.
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ABSTRACT: miRNAs in circulating extracellular vesicles (EVs) are promising biomarkers for cancer. However, their diagnostic ability for early-stage non-small-cell lung cancer (NSCLC) is not well known. In this study, the circulating EV miRNAs profiling was initially performed in 36 untreated NSCLC patients and 36 healthy controls by TaqMan Low Density Array (TLDA). Subsequently, we performed quantitative reverse-transcription PCR assay (RT-qPCR) validation in several independent cohorts that included 159 NSCLC patients, 120 age/sex-matched healthy controls and 31 benign nodule patients enrolled from three different clinical centres. In addition, 38 cases of NSCLC were analysed before and after surgery. We demonstrated that miR-520c-3p and miR-1274b were significantly and steadily increased in NSCLC patients in comparison with healthy controls and benign nodule patients (P < 0.001) and decreased markedly after tumour resection (P < 0.001). The areas under the curve (AUCs) of the ROC curve of the two-miRNA panel were 0.857 (95% CI, 0813-0.901; P < 0.0001) and 0.845 (95% CI, 0.793-0.896; P < 0.0001) for NSCLC and NSCLC stage I, respectively. Furthermore, the panel was able to differentiate NSCLC from benign nodules with an AUC of 0.823 (95% CI, 0.730-0.915; P < 0.0001). Furthermore, logistic regression analysis revealed the two-miRNA panel as an independent risk factor for NSCLC (OR = 16.128, P < 0.0001). In conclusion, miR-520c-3p and miR-1274b have biomarker potential for early diagnosis of NSCLC in multiple centres.
Project description:BackgroundBiomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC.MethodsUnstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel.ResultsWe identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93).ConclusionsWe have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.
Project description:The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.
Project description:The aim of the study was a search for diagnostic and/or prognostic biomarkers in patients with non-small cell lung cancer (NSCLC) patients, based on circulating microRNAs (miRs: miR-23a, miR-361, miR-1228 and miR-let7i) in extracellular vesicles (EVs). Serum EVs were isolated from NSCLC patients (n = 31) and control subjects (n = 21). RNA was isolated from EVs and reverse transcription reaction was performed. Relative levels of miR-23a, miR-361, miR-1228 and miR-let7i were assessed in real-time qPCR using TaqMan probes. Analysis was based on the 2-ΔΔCT method. Statistically significant lower levels of miR-23a and miR-let7i were observed among NSCLC patients vs. control group: miR-23a, 0.054 vs. 0.107; miR-let7i, 0.193 vs. 0.369 (p = 0.003, p = 0.005, respectively). A receiver operating characteristic (ROC) curve analysis demonstrated the diagnostic potential of each individual serum EV-derived miRNA with an area under the curve AUC = 0.744 for miR-23a (p = 0.0003), 0.733 for miR-let7i (p = 0.0007). The decreased level of miR-23a in patients correlated with metastasis to lymph nodes and with AJCC tumor staging system. The results demonstrate that miR-23a and miR-let7i may prove clinically useful as significant, non-invasive markers in NSCLC diagnosis. Additionally, changing profile level of miR-23a that correlates with cancer development may be considered as an NSCLC progression marker.
Project description:BackgroundSeveral studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs.MethodsOSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves.ResultsA preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC.ConclusionsIn this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.
Project description:It takes more than 20 years for normal colorectal mucosa to develop into metastatic carcinoma. The long time window provides a golden opportunity for early detection to terminate the malignant progression. Here, we aim to enable liquid biopsy of T1a stage colorectal cancer (CRC) and precancerous advanced adenoma (AA) by profiling circulating small extracellular vesicle (sEV)-derived RNAs. We exhibited a full RNA landscape for the circulating sEVs isolated from 60 participants. A total of 58,333 annotated RNAs were detected from plasma sEVs, among which 1,615 and 888 sEV-RNAs were found differentially expressed in plasma from T1a stage CRC and AA compared to normal controls (NC). Then we further categorized these sEV-RNAs into six modules by a weighted gene coexpression network analysis and constructed a 60-gene t-SNE model consisting of the top 10 RNAs of each module that could well distinguish T1a stage CRC/AA from NC samples. Some sEV-RNAs were also identified as indicators of specific endoscopic and morphological features of different colorectal lesions. The top-ranked biomarkers were further verified by RT-qPCR, proving that these candidate sEV-RNAs successfully identified T1a stage CRC/AA from NC in another cohort of 124 participants. Finally, we adopted different algorithms to improve the performance of RT-qPCR-based models and successfully constructed an optimized classifier with 79.3% specificity and 99.0% sensitivity. In conclusion, circulating sEVs of T1a stage CRC and AA patients have distinct RNA profiles, which successfully enable the detection of both T1a stage CRC and AA via liquid biopsy.
Project description:Immune disorders caused by sepsis have recently drawn much attention. We sought to dynamically monitor the expression of small extracellular vesicle (sEV) miRNAs in peripheral blood during sepsis to explore these miRNAs as potential biomarkers for monitoring immune function in sepsis patients. This study included patients with sepsis. Blood samples were obtained from 10 patients on the first through 10th days, the 12th day and the 14th day since sepsis onset, resulting in 120 collected samples. Serum sEVs were extracted from peripheral venous blood, and levels of MIR497HG, miR-195, miR-497, and PD-L1 in serum sEVs were detected by qPCR, and clinical information was recorded. Our study revealed that the levels of MIR497HG, miR-195, miR-497 and PD-L1 in serum sEVs showed periodic changes; the time from peak to trough was approximately 4-5 days. The levels of sEV MIR497HG and miR-195 had a positive linear relationship with SOFA score (r values were -0.181 and -0.189; p values were 0.048 and 0.039, respectively). The recorded quantities of sEV MIR497HG, miR-195 and PD-L1 showed a substantial correlation with ARDS. ROC curve analysis revealed that sEV MIR497HG, miR-195 and miR-497 could predict the 28-day mortality of sepsis patients with an AUC of 0.66, 0.68 and 0.72, respectively. Levels of sEVs MIR497HG, miR-195, miR-497 and PD-L1 showed periodic changes with the immune status of sepsis, which provides a new exploration direction for immune function biomarkers and immunotherapy timing in sepsis patients.
Project description:Extracellular vesicles (EVs) play a vital role in normal lung physiology to maintain homeostasis in the airways via intercellular communication. EVs include exosomes and microvesicles, and are characterized by their phospholipid bilayers. EVs have been recognized as novel circulating biomarkers of disease, which are released by different cell types. In this study, we used different EV isolation and purification methods to characterize the plasma-derived EV miRNAs from non-smokers, smokers and patients with COPD. A small RNA sequencing (RNA-seq) approach was adapted to identify novel circulating EV miRNAs. We found that plasma-derived EVs from non-smokers, smokers and patients with COPD vary in their size, concentration, distribution and phenotypic characteristics as confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of EV surface markers. RNA-seq analysis confirmed the most abundant types of small RNAs, such as miRNAs, tRNAs, piRNAs snRNAs, snoRNAs and other biotypes in plasma-derived EVs. We mainly focused on miRNAs as novel biomarkers in smokers and patients with COPD for further analysis. Differential expression by DESeq2 identified distinct miRNA profiles (up-regulated: miR-22-3p, miR-99a-5p, miR-151a-5p, miR-320b, miR-320d; and down-regulated: miR-335-5p, miR-628-3p, miR-887-5p and miR-937-3p) in COPD versus smokers or non-smokers in a pairwise comparison. Gene set enrichment analysis (GSEA) of differentially expressed miRNAs revealed the top pathways, gene ontology and diseases associated with smokers and patients with COPD. We selectively validated miRNAs in EVs isolated from BEAS-2B cells treated with cigarette smoke extract by quantitative PCR analysis. For the first time, we report that plasma-derived EV miRNAs are novel circulating pulmonary disease biomarkers. Thus, molecular profiling of EV miRNAs has great translational potential for the development of biomarkers that may be used in the diagnosis, prognosis, and therapeutics of COPD.
Project description:The objective of this study was to analyze the extracellular vesicle microRNA (EV-miRNA) expression profiles in the blood plasma of heifers with variable response to superstimulation.
Project description:The aim of this study was to investigate the miRNA profiles of nanosized small extracellular vesicles (sEVs) from human retinal pigment epithelial (RPE) cells under oxidative damage. ARPE-19 cells were cultured with ox-LDL (100 mg/L) or serum-free medium for 48 hours, sEVs were then extracted, and miRNA sequencing was conducted to identify the differentially expressed genes (DEGs) between the 2 groups. RNA sequence results were validated using quantitative real-time PCR. The Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes pathway, and ingenuity pathway analyses (IPA) were performed for the DEGs. Results revealed that oxidative stress inhibited RPE cell viability and promoted sEV secretion. A total of 877 DEGs from sEVs were identified, of which 272 were downregulated and 605 were upregulated. In total, 66 enriched GO terms showed that the 3 most significant enrichment terms were cellular processes (biological processes), cell (cellular component), and catalytic activity (molecular function). IPA were used to explore DEGs associated with oxidation damage and further construct a miRNA-target regulatory network. This study identified several DEGs from oxidation-stimulated RPE cells, which may act as potential RNA targets for prognosis and diagnosis of RPE degeneration.
Project description:Circular microRNAs (miRNAs) have become central in pathophysiological conditions of atherosclerosis (AS). However, the biomarkers for diagnosis and therapeutics against AS are still unclear. The atherosclerosis models in low-density lipoprotein receptor deficiency (LDLr-/-) mice were established with a high-fat diet (HFD). The extraction kit isolated extracellular vesicles from plasma. Total RNAs were extracted from LDLr-/- mice in plasma extracellular vesicles. Significantly varying miRNAs were detected by employing Illumina HiSeq 2000 deep sequencing technology. Target gene predictions of miRNAs were employed by related software that include RNAhybrid, TargetScan, miRanda, and PITA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) further analyzed the intersection points of predicted results. The results showed that the HFD group gradually formed atherosclerotic plaques in thoracic aorta compared with the control group. Out of 17, 8 upregulated and 9 downregulated miRNAs with a significant difference were found in the plasma extracellular vesicles that were further cross-examined by sequencing and bioinformatics analysis. Focal adhesion and Ras signaling pathway were found to be the most closely related pathways through GO and KEGG pathway analyses. The 8 most differentially expressed up- and downregulated miRNAs were further ascertained by TaqMan-based qRT-PCR. TaqMan-based qRT-PCR and in situ hybridization further validated the most differentially expressed miRNAs (miR-378d, miR-181b-5p, miR-146a-5p, miR-421-3p, miR-350-3p, and miR-184-3p) that were consistent with deep sequencing analysis suggesting a promising potential of utility to serve as diagnostic biomarkers against AS. The study gives a comprehensive profile of circular miRNAs in atherosclerosis and may pave the way for identifying biomarkers and novel targets for atherosclerosis.