Project description:Exosomal miRNAs have been studied in relation to many diseases. However, there is little to no knowledge regarding the miRNA population of BALF or the lung tissue derived exosomes in COPD and IPF. Considering this, we determined and compared the miRNA profiles of BALF and lung tissue-derived exosomes from healthy non-smokers, healthy smokers, and patients with COPD and IPF. NGS results identified three differentially expressed miRNAs in the BALF, while one in the lung-derived exosomes from COPD patients as compared to healthy non-smokers. Of these, we found three- and five-fold downregulation of miR-122-5p amongst the lung tissue-derived exosomes from COPD patients as compared to healthy non-smokers and smokers, respectively. Interestingly, there were key 55 differentially expressed miRNAs in the lung tissue-derived exosomes of IPF patients compared to non-smoking controls.
Project description:Abstract: Background: MiRNA signatures in human sera have been reported for various tumor diseases. Here we generated miRNA profiles analyzing 1205 mature miRNA transcripts of serum samples of Wilms tumor patients, taken prior and after chemotherapy according to SIOP protocol 2001. Using a feature subset selection filter approach we identified a minimal number of miRNAs with a maximum contribution for the classification between treated and untreated patients and between patients and controls. Results: Analyzing 1205 mature miRNAs, we separated controls and Wilms tumor patients prior chemotherapy with an accuracy of 0.81. We obtained a similar accuracy (0.82) for the separation between controls and sera of Wilms tumor patients after preoperative chemotherapy. We identified 23 miRNAs that were differentially expressed in both comparisons. Subset selection improved the overall classification accuracy between controls and Wilms tumor patients prior and after chemotherapy to 0.94 and 0.91, respectively. Subset selection also allowed separating between Wilms tumor patients prior and after chemotherapy with an accuracy of 0.98. Conclusion: Our analysis identified serum based miRNA signatures that allowed separating between controls, untreated Wilms tumor patients, and Wilms tumor patients after chemotherapy with high accuracy for each of these comparisons.
Project description:RNA was isolated from serum of pateints with PAS, CC and healthy controls. Global microRNA profiling was done using miRNome microRNA Profiler QuantiMir Human PCR Array (#RA660A-1, version 15; BioCat, Heidelberg, Germany) miRNA arrays were performed on pooled RNA samples from six patients for PSC and CC each and a comparison was performed to healthy control patients. MiRNAs with Ct value above 32 were discarded.
Project description:Cushing’s syndrome (CS) is a rare disease with high morbidity and mortality. Diagnosis and subtyping are complex and challenging. Circulating microRNAs were described to be useful as minimally invasive diagnostic markers. Our aim was to determine and compare the circulating microRNA expression profiles of patients with CS and controls. We included three groups of patients of the German Cushing’s registry: A.) patients with florid adrenal dependent CS scheduled for adrenalectomy (CPA); B.) patients with florid pituitary dependent CS scheduled for surgery (CD); and C.) patients in whom CS had been ruled out (controls). Next-generation sequencing was performed in the 30 CS serum samples before and after curative surgery, respectively, and in 10 baseline samples of controls. No significant differential expression was observed between all the CS samples and controls by NGS as well as by QPCR. The sequencing of the preoperative samples revealed a significant differential expression of miR-182-5p (p=0.02) between CD and controls. The differential expression was validated by QPCR in the discovery cohort and in an independent validation cohort. MiR-96-5p, miR-146b-5p, miR-183-5p, miR-185-5p, miR-616-5p and miR-629-5p were found to be significantly upregulated in CPA samples in comparison to CD group of the preoperative group in NGS. However, similar upregulation pattern could not be observed by QPCR in the discovery and validation cohorts. Additionally, miR-96-5p and miR-185-5p were found to be modulated by dexamethasone in controls. Thus, our study reports miR-182-5p as a possible biomarker for CD, which has to be validated in a prospective cohort.
Project description:This experiment was carried out to see if there were any miRNA expression differences in pulmonary endothelial cells between patients with and without COPD. COPD is an inflammatory condition and although much work has previously been performed to investigate the inflammatory cells in COPD there has not been as much research looking at the endothelium through which inflammatory cells must pass through to reach the lung tissue. In this experiment pulmonary endothelial cells were extracted from whole lung tissue removed at the time of cardiothoracic surgery. This was performed for patients with and without COPD. RNA was extracted using the Qiagen microRNeasy kits prior to transferring to the University of Birmingham Biosciences department who performed RNA labelling and ran the microarrays. Once the microarrays were performed quality was checked using ArrayQualityMetrics and the COPD group was compared to the non-COPD group using SAM. The experiment was then repeated using another patient group. MiRNAs of interest were validated with qPCR initially before moving on to functional work.
Project description:Radiation therapy is an effective cancer treatment although damage to healthy tissues is common. Here we establish sequencing-based, cell-type specific DNA methylation reference maps of human and mouse tissues to infer the origins of cell-free DNA fragments released from dying cells into the circulation. We find cell-type specific DNA blocks mostly hypomethylated and located within genes intrinsic to cellular identity. In a mouse model, thoracic radiation-induced tissue damages were reflected by dose-dependent increases in lung endothelial, cardiomyocyte and hepatocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, cell-free methylated DNA in serum can uncover cell-type specific effects of radiation on healthy tissues and inform treatment.
Project description:Radiation therapy is an effective cancer treatment although damage to healthy tissues is common. Here we establish sequencing-based, cell-type specific DNA methylation reference maps of human and mouse tissues to infer the origins of cell-free DNA fragments released from dying cells into the circulation. We find cell-type specific DNA blocks mostly hypomethylated and located within genes intrinsic to cellular identity. In a mouse model, thoracic radiation-induced tissue damages were reflected by dose-dependent increases in lung endothelial, cardiomyocyte and hepatocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, cell-free methylated DNA in serum can uncover cell-type specific effects of radiation on healthy tissues and inform treatment.
Project description:Lung cancer (LC) is one of the major cancers, with survival of patients dictated by the time of diagnosis. Cell-free circulating miRNAs have been proposed as candidate biomarkers for non-small cell lung cancer. The goal of this study was to profile the miRNAs in blood plasma of lung cancer patients diagnosed with squamous cell carcinoma (SCC, n=14) or adenocarcinoma (AD, n=6), and healthy individuals (HD) using miRCURY LNA miRNA qPCR Serum/Plasma Panel (Exiqon). Average Cq of detected assays was used for normalization of miRNA expression.
Project description:COPD is a heterogeneous condition without effective disease modifying therapies. Identification of novel inflammatory endotype markers such as extracellular vesicles (EVs), which are important intercellular messengers carrying microRNA (miRNA), may enable earlier diagnosis and disease stratification for a targeted treatment approach. Our aim was to identify differentially expressed EV miRNA in the lungs of COPD patients compared with healthy ex-smokers and determine whether they can help define inflammatory COPD endotypes. EV miRNA were isolated and sequenced from ex-smoking COPD patients and healthy ex-smoker bronchoalveolar lavage fluid. Results were validated with RT-qPCR and compared to differential inflammatory cell counts. Differential expression analysis identified five upregulated miRNA in COPD (miR-223-3p, miR-2110, miR-182-5p, miR-200b-5p and miR-625-3p) and three downregulated miRNA (miR-138-5p, miR-338-3p and miR-204-5p), all with a log2 fold change of >1/-1, FDR<0.05. These miRNAs correlated with disease defining characteristics such as FEF 25-75% (a small airways disease measure) and DLCO % (a surrogate measure of emphysema). Receiver operator curve analysis demonstrated miR-2110, miR-223-3p and miR-182-5p showed excellent combinatory predictive ability (AUC 0.91, p<0.0001) in differentiating between health and mild COPD. Furthermore, miR-223-3p and miR-338-3p correlated with airway eosinophilia and were able to distinguish “pure eosinophilic” COPD from other airway inflammatory subtypes (AUC 0.94 and 0.85 respectively). This is the first study to identify differentially expressed miRNA in COPD bronchoalveolar lavage fluid EVs. These findings suggest specific lung derived EV miRNA are a strong predictor of disease presence even in mild COPD. Furthermore, specific miRNA correlated with inflammatory cell numbers in COPD, and may have a role in defining inflammatory endotypes for future treatment stratification.
Project description:The aim of this study was to explore novel miRNA biomarkers in the serum of lung cancer patients based on miRNA profiling. A miRCURY™ LNA Array was used to identify differentially altered miRNAs in the serum of lung cancer patients (n = 6) and healthy controls (n = 6). Total RNA was isolated using TRI reagent BD (MRCgene, TB-126) according to manufacturer’s instructions. After having passed RNA quantity measurement using the NanoDrop 1000, the samples were labeled using the miRCURY™ Hy3™/Hy5™ Power labeling kit and hybridized on the miRCURY™ LNA Array (v.18.0). Following the washing steps the slides were scanned using the Axon GenePix 4000B microarray scanner.Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs were identified through Volcano Plot filtering. Finally, hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples.