Differential expression of exosomal miRNAs between breast cancer patients with and without recurrence.
ABSTRACT: Exosomal microRNAs (miRNAs) are promising candidate biomarkers for diagnosis or prognosis for breast cancer. We investigated the prognostic role of exosomal miRNAs in serum samples derived from patients with breast cancer and compared miRNA expression between serum and tumor tissues.The miRNA profile derived from exosome between breast cancer patients with recurrence (n = 16) and without recurrence (n = 16) were compared by miRNA PCR array. Further, we examined the expression of miRNAs derived from tissues in the patients with breast cancer with (n = 35) and without recurrence (n = 39) by qRT-PCR.Of 384 miRNAs, three miRNAs (miR-338-3p, miR-340-5p, and miR-124-3p) were significantly upregulated and eight (miR-29b-3p, miR-20b-5p, miR-17-5p, miR-130a-3p, miR-18a-5p, miR-195-5p, miR-486-5p, and miR-93-5p) were significantly downregulated in the patients with recurrence. We evaluated the expression of the miRNAs in tumor tissues. The patients with recurrence had higher levels of miR-340 at their primary site as well as in the serum. In contrast, miR-195-5p, miR-17-5p, miR-93-5p, and miR-130a-3p, derived from tumor tissues that were downregulated in the serum from patients with recurrence, were higher in the patients with recurrence than in those with no recurrence. In logistic regression analysis, miR-340-5p, miR-17-5p, miR-130a-3p, and miR-93-5p were significantly associated with recurrence.Several exosomal miRNAs may be useful biomarkers to predict breast cancer recurrence. We show the different expression patterns of miRNAs between tumor tissues and serum. These findings may suggest selective mechanism of release of exosomal miRNAs by cancer cells to regulate their progression.
Project description:Novel noninvasive biomarkers with high sensitivity and specificity for the diagnosis of breast cancer (BC) are urgently needed in clinics. The aim of this study was to explore whether miRNAs from the miR-106a-363 cluster can be detected in the circulation of BC patients and whether these miRNAs can serve as potential diagnostic biomarkers.The expression of 12 miRNAs from the miR-106a-363 cluster was evaluated using qRT-PCR in 400 plasma samples (from 200 BC patients and 200 healthy controls (HCs)) and 406 serum samples (from 204 BC patients and 202 HCs) via a three-phase study. The identified miRNAs were further examined in tissues (32 paired breast tissues), plasma exosomes (from 32 BC patients and 32 HCs), and serum exosomes (from 32 BC patients and 32 HCs).Upregulated levels of four plasma miRNAs (miR-106a-3p, miR-106a-5p, miR-20b-5p, and miR-92a-2-5p) and four serum miRNAs (miR-106a-5p, miR-19b-3p, miR-20b-5p, and miR-92a-3p) were identified and validated in BC. A plasma 4-miRNA panel and a serum 4-miRNA panel were constructed to discriminate BC patients from HCs. The areas under the receiver-operating characteristic curves of the plasma panel were 0.880, 0.902, and 0.858, and those of the serum panel were 0.910, 0.974, and 0.949 for the training, testing, and external validation phases, respectively. Two overlapping miRNAs (miR-106a-5p and miR-20b-5p) were consistently upregulated in BC tissues. Except for the expression of the plasma-derived exosomal miR-20b-5p, the expression patterns of exosomal miRNAs were concordant between plasma and serum, indicating the potential use of exosomal miRNAs as biomarkers.We identified four plasma miRNAs and four serum miRNAs from the miR-106a-363 cluster as promising novel biomarkers for the diagnosis of BC.
Project description:BACKGROUND:MicroRNAs (miRNAs) play an important role in the development and progression of breast cancer (BC). The purpose of the present study was to identify plasma miRNAs enabling early diagnosis of BC. MATERIALS AND METHODS:Expression levels of seven plasma miRNAs (miR-23a-3p, miR-29b-2-5p, miR-130a-5p, miR-144-3p, miR-148a-3p, miR-152-3p, and miR-182-5p) in 106 patients with newly diagnosed BC and 96 healthy participants were analyzed by qRT-PCR. We also evaluated the relationship between the expression levels of these miRNAs and clinicopathological features of patients with BC. RESULTS:Compared with healthy controls, we found that miR-23a-3p (p = .025), miR-130a-5p (p = .006), miR-144-3p (p = .040), miR-148a-3p (p = .023), and miR-152-3p (p = .019) were downregulated in the plasma of patients with BC. MiR-130a-5p, miR-144-3p, and miR-152-3p were downexpressed in BC tissues as well as plasma. The expression of the miR-23a-3p, miR-144-3p, and miR-152-3p was related to ER positive and PR positive. Besides, miR-23a-3p, miR-144-3p, and miR-152-3p did show the significant difference in the staging compromised to the control, especially in stage I-II. Moreover, we also found that miR-144-3p and miR-148a-3p were associated with lymph node invasion. CONCLUSIONS:The expression levels of the miR-23a-3p, miR-130a-5p, miR-144-3p, miR-148a-3p, and miR-152-3p were lower in patients with BC compared to healthy controls and were associated with ex hormone receptor, clinical stage, and lymph node metastasis, indicating the diagnostic potential of these miRNAs in BC.
Project description:Gastric cancer (GC) remains a leading cause of cancer-related mortality in the United States and China, there is an urgent need to discover novel non-invasive biomarkers for the early diagnosis of GC to improve the prognosis of GC patients. Exosomal miRNAs are considered promising biomarkers for cancer diagnosis. Using next-generation sequencing (NGS), bioinformatics and further validation, we identified and evaluated exosomal miRNAs in serum as early diagnostic markers for GC. NGS revealed that the average mappable reads in the RNA libraries were about 6.5 million per patient including miRNAs (73.38%), rRNAs (17.10%), snRNAs (8.83%), snoRNAs (0.65%), and tRNAs (0.04%). A total of 66 up and 13 down-regulated exosomal miRNAs were found in the screened cohort. In the validation cohort, by comparing with healthy individuals, higher levels of serum exosomal miR-92b-3p, let-7g-5p, miR-146b-5p, and miR-9-5p were found to be significantly associated with early-stage GC (p < 0.05). Diagnostic power of the combined panels of the exosomal miRNAs or the combination of exosomal miRNAs and CEA outperformed that of single exosomal miRNA marker for establishing a diagnosis of early-stage GC. The combined diagnosis of exosomal miR-92b-3p + let-7g-5p + miR-146b-5p + miR-9-5p with CEA had the most powerful efficiency with an AUC up to 0.786. In addition, serum levels of exosomal miR-92b-3p were significantly associated with poor cohesiveness (p = 0.0021), let-7g-5p and miR-146b-5p were significantly correlated with nerve infiltration (p = 0.0234 and p = 0.0126, respectively), and miR146b-5p was statistically correlated with tumor invasion depth in early-stage GC (p = 0.0089). In conclusion, serum exosomal miR-92b-3p, -146b-5p, -9-5p, and let-7g-5p may serve as potential non-invasive biomarkers for early diagnosis of GC.
Project description:OBJECTIVE: Triple-negative breast cancer (TNBC) patients with truly chemosensitive disease still represent a minority among all TNBC patients. The aim of the present study is to identify microRNAs (miRNAs) that correlate with TNBC chemoresistance. METHODS: In this study, we conducted miRNAs profile comparison between triple-negative breast cancer (TNBCs) and normal breast tissues by microRNA array. Quantitative real-time PCR (qRT-PCR) was utilized to confirm the specific deregulated miRNAs change trend. We used starBase 2.1 and GOrilla to predict the potential targets of the specific miRNAs. Cells viability and apoptosis assays were employed to determine the effect of alteration of the specific miRNAs in TNBC cells on the chemosensitivity. RESULTS: We identified 11 specific deregulated miRNAs, including 5 up-regulated miRNAs (miR-155-5p, miR-21-3p, miR-181a-5p, miR-181b-5p, and miR-183-5p) and 6 down-regulated miRNAs (miR-10b-5p, miR-451a, miR-125b-5p, miR-31-5p, miR-195-5p and miR-130a-3p). Thereafter, this result was confirmed by qRT-PCR. We predicted the potential targets of the candidate miRNAs and found that they are involved in cancer-associated pathways. For the first time, we found that miR-130a-3p and miR-451a were down-regulated in TNBC. 9 of the 11 specific deregulated miRNAs were found to be associated with chemoresistance. In vitro assays, we found that up-regulation of either miR-130a-3p or miR-451a in MDA-MB-231 cells significantly changed the cells sensitivity to doxorubicin. The results suggest that TNBC chemotherapy might be affected by a cluster of miRNAs. CONCLUSION: The abnormal expression miRNAs in TNBC are mainly chemoresistance related. This might be part of reason that TNBC likely to evade from chemotherapy resulting in early relapse and high risk of death. To alter their expression status might be a potential therapeutic strategy to improve the outcome of chemotherapy for TNBC patients.
Project description:Background:A reliable noninvasive biomarker is not yet available for endometriosis diagnosis. Novel biomarkers for the diagnosis of endometriosis are urgently needed. The molecular constituents of exosomes, especially exosomal microRNAs (miRNAs), have considerable potential as novel biomarkers for clinical diagnosis. This study is aimed at exploring aberrant exosomal miRNA profiles by using miRNA microarray and at providing more accurate molecular biomarkers of endometriosis. Methods:Exosomes were isolated from the serum of patients with endometriosis and negative controls and identified by electron microscopy, nanoparticle tracking analysis, and Western blot. Exosomal miRNAs were profiled by miRNA microarrays. The expression of selective serum exosomal miRNA was validated by qRT-PCR. Receiver operating characteristic (ROC) curves were established to explore the diagnostic value of selective miRNAs. Finally, GO annotation and KEGG pathway enrichment analyses were used to display possible functions associated with the two miRNAs. Results:A total of 24 miRNAs showed differential levels of enrichment with P < 0.05 and |log2?fold?change| > 1 by miRNA microarrays. Among the six selective miRNAs (i.e., miR-134-5p, miR-197-5p, miR-22-3p, miR-320a, miR-494-3p, and miR-939-5p), qRT-PCR analysis revealed that miR-22-3p and miR-320a were significantly upregulated in serum exosomes from patients with endometriosis compared with negative individuals. ROC curve revealed that the serum exosomal miR-22-3p and miR-320a yielded the area under the curve values of 0.855 and 0.827, respectively. Conclusion:Our results demonstrated that exosomal miR-22-3p and miR-320a were significantly increased in the sera of patients with endometriosis. The two miRNAs may be useful potential biomarkers for endometriosis diagnosis.
Project description:Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). There is currently no single definitive test for MS. Circulating exosomes represent promising candidate biomarkers for a host of human diseases. Exosomes contain RNA, DNA, and proteins, can cross the blood-brain barrier, and are secreted from almost all cell types including cells of the CNS. We hypothesized that serum exosomal miRNAs could present a useful blood-based assay for MS disease detection and monitoring. Exosome-associated microRNAs in serum samples from MS patients (n?=?25) and matched healthy controls (n?=?11) were profiled using small RNA next generation sequencing. We identified differentially expressed exosomal miRNAs in both relapsing-remitting MS (RRMS) (miR-15b-5p, miR-451a, miR-30b-5p, miR-342-3p) and progressive MS patient sera (miR-127-3p, miR-370-3p, miR-409-3p, miR-432-5p) in relation to controls. Critically, we identified a group of nine miRNAs (miR-15b-5p, miR-23a-3p, miR-223-3p, miR-374a-5p, miR-30b-5p, miR-433-3p, miR-485-3p, miR-342-3p, miR-432-5p) that distinguished relapsing-remitting from progressive disease. Eight out of nine miRNAs were validated in an independent group (n?=?11) of progressive MS cases. This is the first demonstration that microRNAs associated with circulating exosomes are informative biomarkers not only for the diagnosis of MS, but in predicting disease subtype with a high degree of accuracy.
Project description:Exosomal miRNAs are currently being explored as a novel class of biomarkers in cardiovascular diseases. However, few reports have focused on the value of circulating exosomal miRNAs as biomarkers for stable coronary artery disease (SCAD). Here, we aimed to investigate whether miRNAs involved in cardiovascular diseases in circulating exosomes could serve as novel diagnostic biomarkers for SCAD. Firstly, the serum exosomes were isolated and purified by the ExoQuick reagent and identified by transmission electron microscopy, western blot, and nanoparticle tracking analysis. Then, the purified exosomes were quantified by measuring the exosome protein concentration and calculating the total protein amount. Next, eight miRNAs involved in cardiovascular diseases, miR-192-5p, miR-148b-3p, miR-125a-3p, miR-942-5p, miR-149-5p, miR-32-5p, miR-144-3p, and miR-142-5p, were quantified in circulating exosomes from the control group (<i>n</i> = 20) and the SCAD group (<i>n</i> = 20) by quantitative real-time polymerase chain reaction (qPCR). Finally, the gene targets of the differentially expressed miRNAs were predicted, and the functions and signaling pathways of these targets were analyzed using an online database. The isolated exosomes had a bilayer membrane with a diameter of about 100?nm and expressed exosomal markers including CD63, Tsg101, and Flotillin but negatively expressed Calnexin. Both the exosome protein concentration and total protein amount exhibited no significant differences between the two groups. The qPCR assay demonstrated that among the eight miRNAs, the expression levels of miR-942-5p, miR-149-5p, and miR-32-5p in the serum exosomes from the SCAD group were significantly higher than that from the control group. And the three miRNAs for SCAD diagnosis exhibited AUC values of 0.693, 0.702, and 0.691, respectively. GO categories and signaling pathways analysis showed that some of the predictive targets of these miRNAs were involved in the pathophysiology processes of SCAD. In conclusion, our findings suggest that serum exosomal miR-942-5p, miR-149-5p, and miR-32-5p may serve as potential diagnostic biomarkers for SCAD.
Project description:BACKGROUND:Extracellular vesicles (EVs) are endogenous membrane vesicles with a diameter of 30-200?nm. It has been reported that hypoxic cancer cells can release numerous EVs to mediate multiple regional and systemic effects in the tumor microenvironment. METHODS:In this study, we used ultracentrifugation to extract EVs secreted by TE-13, an esophageal squamous carcinoma (ESCC) cell line during normoxia and hypoxia and performed high-throughput sequencing to detect exosomal miRNAs. Gene ontology (GO) and KEGG pathway analyses were used to reveal pathways potentially regulated by the miRNAs. RESULTS:A total of 10 810 miRNAs were detected; 50 were significantly upregulated and 34 were significantly downregulated under hypoxic environment. GO analysis identified enrichment of protein binding, regulation of transcription (DNA-templated), and membrane as molecular function, biological process, and cellular component, respectively. KEGG pathway analysis revealed cancer-associated pathways, phospholipase D signaling pathway, autophagy, focal adhesion and AGE-RAGE signaling as the key pathways. Further verification experiment from qRT-PCR indicated that miR-128-3p, miR-140-3p, miR-340-5p, miR-452-5p, miR-769-5p and miR-1304-p5 were significantly upregulated in EVs from hypoxia TE-13 cells while miR-340-5p was significantly upregulated in two other ESCC cells, ECA109 and TE-1. CONCLUSION:This study, for the first time reveals changes in the expression of exosomal miRNAs in hypoxic ESCC cells and these findings will act as a resource to study the hypoxic tumor microenvironment and ESCC EVs.
Project description:Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell of origin, including microRNA. Exosomes released by glioblastoma cross the blood-brain barrier into the peripheral circulation and carry molecular cargo distinct to that of "free-circulating" miRNA. In this pilot study, serum exosomal microRNAs were isolated from glioblastoma (n?=?12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls and to grade II-III (n?=?10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n?=?4) and independent sets of healthy (n?=?9) and non-glioma (n?=?10) controls were used to further test the specificity and predictive power of this unique exosomal microRNA signature. Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p, and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, six iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven miRNA panel was able to correctly classify all specimens in validation cohorts (n?=?23). Also identified were 23 dysregulated miRNAs in IDHMUT gliomas, a partially overlapping yet distinct signature of lower-grade glioma. Serum exosomal miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported "free-circulating" miRNA studies in GBM patients and appear to be superior.
Project description:MicroRNAs (miRNAs/miRs) in exosomes play crucial roles in the onset, progression and metastasis of cancer by regulating the stability of target mRNAs or by inhibiting translation. In the present study, differentially expressed miRNAs were identified in exosomes of 27 breast cancer patients and 3 healthy controls using RNA sequencing. The differentially expressed microRNAs were selected by bioinformatic analysis. Subjects were followed up for 2 years and exosomal miRNA profiles were compared between patients with and without recurrence of breast cancer. A total of 30 complementary DNA libraries were constructed and sequenced and 1,835 miRNAs were detected. There were no significant differences in the expression of miRNAs between the basal?like, human epidermal growth factor receptor?2+, luminal A, luminal B and healthy control (HC) groups. A total of 54 differentially expressed miRNAs were identified in triple?negative breast cancer (TNBC) patients vs. HCs, including 20 upregulated and 34 downregulated miRNAs. The results of the reverse transcription?quantitative PCR were consistent with this. Receiver operating characteristic curve analyses indicated that miR?150?5p [area under the curve (AUC)=0.705, upregulated], miR?576?3p (AUC=0.691, upregulated), miR?4665?5p (AUC=0.681, upregulated) were able to distinguish breast cancer patients with recurrence from those without recurrence. In conclusion, the present results indicated differences in miRNA expression profiles between patients with TNBC and healthy controls. Certain exosomal miRNAs were indicated to have promising predictive value as biomarkers for distinguishing breast cancer with recurrence from non?recurrence, which may be utilized for preventive strategies.