Dataset Information

Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy.

ABSTRACT:

Background

Circulating exosomes from prostate cancer (PCa) patients undergoing radiotherapy are attractive candidate biomarkers for monitoring treatment response. Multiple workflows for isolation and content characterization of exosomes in biofluids have been attempted. We report a protocol to isolate and characterize exosomal miRNAs content and assess radiation-induced changes.

Methods

In this pilot study, we performed targeted exosomal miRNA profiling of 25 serum samples obtained from PCa patients with intermediate- and high-risk disease treated with curative radiotherapy (RT), and controls. Post-treatment blood samples were collected at least 28 days after radiation therapy as a paired follow-up sample. The complete workflow consisted of two phases: I) filtration and polyethylene glycol salt precipitation phase which enriched particles below 200 nm in size followed by characterization using electron microscopy, and II) flow cytometry. Finally, miRNA expression analysis between untreated and treated patient samples was performed using RNA extraction kit, and qRT-PCR.

Results

In our preliminary data, 1 ml of serum from PCa patients showed higher exosomal concentration (3.68E+10) compared to controls (6.07E+08). The overall expression of exosomes after RT was found to be higher compared to untreated samples; the median value changed from 3.68E+10 to 5.40E+10; p?=?0.52. Using electron microscopy, we were able to visualize cup-shaped vesicles with morphology and size compatible with exosomes. The bead-based flow cytometry showed positivity for exosomal tetraspanins surface markers CD63 and CD9. All five miRNAs (hsa-let-7a-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-21-5p, hsa-miR-99b-5p) have been identified in exosomes. Despite overall changes in hsa-let-7a-5p expression after radiation, the difference was significant only in the high-risk group (p?=?0.037). In addition, the radiation response to hsa-miR-21-5p was elevated in the high-risk group compared to the intermediate group (p?=?0.036).

Conclusions

Herewith, we demonstrated a protocol for isolation of serum exosomes and exosomal miRNA amplification. The recovery of exosomal miRNAs and their differential expression after radiation treatment suggests promising biomarker potential that requires further investigation in larger patient cohorts.

SUBMITTER: Malla B

PROVIDER: S-EPMC6090775 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy.

Malla Bijaya B Aebersold Daniel M DM Dal Pra Alan A

Journal of translational medicine 20180813 1

<h4>Background</h4>Circulating exosomes from prostate cancer (PCa) patients undergoing radiotherapy are attractive candidate biomarkers for monitoring treatment response. Multiple workflows for isolation and content characterization of exosomes in biofluids have been attempted. We report a protocol to isolate and characterize exosomal miRNAs content and assess radiation-induced changes.<h4>Methods</h4>In this pilot study, we performed targeted exosomal miRNA profiling of 25 serum samples obtaine ...[more]

PMID: 30103771

Similar Datasets

Project description:PURPOSE:Novel biomarkers for the diagnosis of prostate cancer (PCa) are urgently required. Increasing evidence suggests that exosomal microRNAs (miRNAs or miRs) in serum may be potential noninvasive biomarkers for certain diseases. The objective of the present study was to investigate and assess whether exosomal miR-141 is an effective biomarker for human PCa. METHODS:In the present study, exosomes were isolated from the serum of patients with PCa, patients with benign prostate hyperplasia (BPH), and healthy volunteers. The total RNA was extracted from the exosomes and the level of miR-141 was analyzed by quantitative reverse transcription-polymerase chain reaction. The expression levels of miR-141 were compared between the whole serum and the serum exosomes of the three groups. Subsequently, the relevance of the exosomal expression of miR-141 to the clinicopathological factors in PCa was investigated. RESULTS:The expression of miR-141 was higher in exosomes compared with whole serum (control group, P=0.0003; BPH group, P=0.0016; PCa group, P<0.0001). The level of serum exosomal miR-141 was significantly higher in the patients with PCa compared with the patients with BPH and the healthy controls (3.85-fold, P=0.0007 and 4.06-fold, P=0.0005, respectively). In addition, the expression levels were significantly higher in metastatic PCa compared with localized PCa (P<0.0001). Receiver-operating characteristic curve revealed that the serum exosomal miR-141 yielded an area under the curve of 0.8694, with 80% sensitivity and 87.1% specificity in discriminating patients with metastatic PCa from the patients with localized PCa. CONCLUSION:Serum exosomes may serve as a more suitable material compared with the whole serum for measuring circulating miR-141 levels in patients with PCa. Exosomal miR-141 is upregulated in the serum from patients with PCa compared with patients with BPH or the healthy volunteers, and it may be a useful potential biomarker for the diagnosis of metastatic PCa.

Project description:Colorectal cancer (CRC) is the most common malignant gastrointestinal tumor worldwide. Serum exosomal microRNAs (miRNAs) play a critical role in tumor progression and metastasis. However, the underlying molecular mechanisms are poorly understood.The miRNAs expression profile (GSE39833) was downloaded from Gene Expression Omnibus (GEO) database. GEO2R was applied to screen the differentially expressed miRNAs (DEmiRNAs) between healthy and CRC serum exosome samples. The target genes of DEmiRNAs were predicted by starBase v3.0 online tool. The gene ontology (GO) and Kyoto Encyclopedia of Genomes pathway (KEGG) enrichment analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool. The protein-protein interaction (PPI) network was established by the Search Tool for the Retrieval of Interacting Genes (STRING) visualized using Cytoscape software. Molecular Complex Detection (MCODE) and cytohubba plug-in were used to screen hub genes and gene modules.In total, 102 DEmiRNAs were identified including 67 upregulated and 35 downregulated DEmiRNAs, and 1437 target genes were predicted. GO analysis showed target genes of upregulated DEmiRNAs were significantly enriched in transcription regulation, protein binding, and ubiquitin protein ligase activity. While the target genes of downregulated DEmiRNAs were mainly involved in transcription from RNA polymerase II promoter, SMAD binding, and DNA binding. The KEGG pathway enrichment analyses showed target genes of upregulated DEmiRNAs were significantly enriched in proteoglycans in cancer, microRNAs in cancer, and phosphatidylinositol-3 kinases/Akt (PI3K-Akt) signaling pathway, while target genes of downregulated DEmiRNAs were mainly enriched in transforming growth factor-beta (TGF-beta) signaling pathway and proteoglycans in cancer. The genes of the top 3 modules were mainly enriched in ubiquitin mediated proteolysis, spliceosome, and mRNA surveillance pathway. According to the cytohubba plugin, 37 hub genes were selected, and 4 hub genes including phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), SRC, cell division cycle 42 (CDC42), E1A binding protein p300 (EP300) were identified by combining 8 ranked methods of cytohubba.The study provides a comprehensive analysis of exosomal DEmiRNAs and target genes regulatory network in CRC, which can better understand the roles of exosomal miRNAs in the development of CRC. However, these findings require further experimental validation in future studies.

Project description:BACKGROUND:Adjuvant (ART) and salvage radiotherapy (SRT) are two common concepts to enhance biochemical relapse free survival (BCRFS) in patients with prostate cancer (PC). We analyzed differences in outcome between ART and SRT in patients with steep decline of PSA-levels after surgery to compare outcome. METHODS:We evaluated 253 patients treated with postoperative RT with a median age of 66?years (range 42-85?years) treated between 2004 and 2014. Patients with additive radiotherapy due to PSA persistence and patients in the SRT group, who did not achieve a postoperative PSA level?<0.1?ng/mL were excluded. Hence, data of 179 patients was evaluated. We used propensity score matching to build homogenous groups. A Cox regression model was used to determine differences between treatment options. Median follow-up was 32.5?months (range 1.4-128.0?months). RESULTS:Early SRT at PSA levels <0.3?ng/mL was associated with significant longer BCRFS than late SRT (HR: 0.32, 95%-CI: 0.14-0.75, p =?0.009). Multiple Cox regression showed pre-RT PSA level, tumor stage, and Gleason score as predictive factors for biochemical relapse. In the overall group, patients treated with either ART or early SRT showed no significant difference in BCRFS (HR: 0.17, 95%-CI: 0.02-1.44, p =?0.1). In patients with locally advanced PC (pT3/4) BCRFS was similar in both groups as well (HR: 0.21, 95%-CI:0.02-1.79, p =?0.15). CONCLUSION:For patients with PSA-triggered follow-up, close observation is essential and early initiation of local treatment at low PSA levels (<0.3?ng/mL) is beneficial. Our data suggest, that SRT administered at early PSA rise might be equieffective to postoperative ART in patients with locally advanced PC. However, the individual treatment decision must be based on any adverse risk factors and the patients' postoperative clinical condition. STUDY REGISTRATION:The present work is approved by the Ethics Commission of the Technical University of Munich (TUM) and is registered with the project number 320/14.

Dataset Information

Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy.

Background

Methods

Results

Conclusions

Publications

Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure