Detecting lung tumors from circulating microRNA profiles
ABSTRACT: We aimed to determine if lung cancer detection can be improved by circulating miRNAs as biomarkers. To this end, we collected blood of over 3000 individuals using PAXgene blood tubes. The individuals were diagnosed with lung cancer (LCa), a non-tumor lung disease (NTLD), another disease (OD) or were healthy controls. For every individual we determined their miRNA expression patterns. Total RNA was extracted with Qiagen PAXgene Blood miRNA Kit, labeled and hybridized with the Agilent miRNA Complete Labeling and Hyb Kit and scanned with Agilent microarray scanner system.
Project description:Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays. Blood from 6 healthy individuals was collected in one PAXgeneTM blood RNA tube (Becton Dickinson, 2.5 ml blood) and one dipotassium EDTA blood tube (EDTA-KE Monovette, Sarstedt, 9 ml blood) per individual. There was no known disease for any of the blood donors. A fixed volume of 2.5 ml blood from the EDTA tube was transferred at three different time points after blood withdrawal (0 min, 10 min, and 2 h) into fresh PAXgene blood RNA tubes to ensure stabilization of the RNA in the blood samples. All PAXgene blood tubes were stored at room temperature until at least 2 h after the last transfer of EDTA blood into PAXgene tubes, to ensure complete lysis of the blood cells, before they were stored at -20°C until RNA isolation.
Project description:Background: There is ample evidence of blood-born miRNA signatures for various human diseases. To dissect the origin of disease-specific miRNA expression in human blood cells, we separately analyzed the miRNome of eosinophilic and neutrophilic granulocytes, monocytes, B-cells, T-cells, and natural killer cells, each in lung cancer patients and healthy individuals. Results: We found specific miRNA expression patterns for each immune cell type and also depending on the cell origin, line of defense, and function. The overall expression pattern of each leukocyte subtype showed great similarities between lung cancer patients and healthy controls. However, for each cell subtype we identified miRNAs that were deregulated in lung cancer patients including hsa-miR-21, a well- known oncomiR associated with poor lung cancer prognosis that was up-regulated in all subtype comparisons of lung cancer versus controls. While the miRNome of cells of the adaptive immune system allowed only a weak separation between patients and controls, cells of the innate immune system allowed perfect or nearly perfect classification. Conclusions: Leukocytes of lung cancer patients show a cancer-specific miRNA expression profile. Our data also show that cancer specific miRNA expression pattern of whole blood samples are not determined by a single cell type. The data indicate that additional blood components, like erythrocytes, platelets, or exosomes might contribute to the disease specificity of a miRNA signature. Overall design: We obtained EDTA and PAXgene blood from 7 healthy subjects and 7 lung cancer patients and isolated the CD3, CD19, CD15, CD14, and CD56 positive cell fractions from the EDTA blood samples to compare the miRNomes of the cell subsets of healthy individuals versus lung cancer patients.
Project description:To determine the miRNA expression pattern in RBCs, we isolated miRNA from RBCs of healthy blood donors, who were homozygous or heterozygous carriers of different AB0 blood groups. We first analyzed miRNA expression by microarray chip analysis. In average, we found 873 miRNAs to be present in RBCs with a partially differential expression pattern depending on the blood group genotype. 148 out of these 873 miRNAs were significantly up- or downregulated in RBCs of blood group 0 and of heterozygous genotypes, as compared to homozygous genotypes. For each blood group genotype erythrocyte concentrates of three different donors were used for microarray analysis.
Project description:Background: MiRNAs from body fluids gain more and more attraction as biomarker candidates. Besides serum, patterns from whole blood are increasingly considered as markers for human pathologies. Usually, the contribution of different cell types to the respective signature remains however unknown. In this study we provide insights into the human miRNome of different compounds of the blood including CD3, CD14, CD15, CD19, CD56 positive cells as well as exosomes. Methods: We measured the miRNA repertoire for each cell type and whole blood for two individuals at three time points over the course of one year in order to provide evidence that the cell type miRNomes can be reproducibly detected. Results: For measurements repeated after 24 hours we found on average correlation of 0.97, even after one year profiles still correlated with 0.96, demonstrating the enormous stability of the cell type specific miRNomes. Highest correlation was found for CD15 positive cells, exceeding Pearson correlation of 0.99. For exosomes a significantly higher variability of miRNA expression was detected. In order to estimate the complexity and variability of the cell type specific miRNomes, we generated profiles for all considered cell types in a total of seven unaffected individuals. While CD15 positive cells showed the most complex miRNome consisting of 328 miRNAs, we detected significantly less miRNAs (186, p = 1.5*10-5) in CD19 positive cells. Moreover, our analysis showed functional enrichment in many relevant categories such as onco-miRNAs and tumor miRNA suppressors. Interestingly, exosomes were enriched just for onco-miRNAs but not for miRNA tumor suppressors. Conclusion: In sum, our results provide evidence that blood cell type specific miRNomes are very consistent between individuals and over time. We obtained EDTA and PAXgene blood from 7 healthy subjects and isolated the CD3, CD19, CD15, CD14, and CD56 positive cell fractions from the EDTA blood samples (timepoint t0). In addition, we repeated the experiment one year later (t1) and one year and a day later (t2) for two samples.
Project description:Identification of renal cortex microRNAs whose expression differs between male individuals with high blood pressure and normal blood pressure using Agilent Human miRNA Microarrays (V3, release 12.0). The Silesian Renal Tissue Bank, a collection of tissues which aimed to investigate candidate genes in human cardiovascular disease, was used to analyze the miRNA expression in hypertensive and normotensive patients. Approximately 1 cm3 of tissue from the healthy (unaffected by cancer) pole of the kidney was obtained immediately after surgery and transferred into containers with RNAlater (Ambion) and preserved at 70°C before mRNA extraction, which used a commercially available assay (RNeasy, Qiagen). Medulla and cortex were separated and individual RNA was obtained for each of them. No pooling was performed. After extraction of RNA, cRNA was prepared and arrays performed using Agilent Human miRNA Microarrays (V3, release 12.0) performed at the Ramaciotti Gene Function Analysis facility, University of New South Wales in Sydney, Australia.
Project description:Identification of renal medulla microRNAs whose expression differs between male individuals with high blood pressure and normal blood pressure using Agilent Human miRNA Microarrays (V3, release 12.0). The Silesian Renal Tissue Bank, a collection of tissues which aimed to investigate candidate genes in human cardiovascular disease, was used to analyze the miRNA expression in hypertensive and normotensive patients. Approximately 1 cm3 of tissue from the healthy (unaffected by cancer) pole of the kidney was obtained immediately after surgery and transferred into containers with RNAlater (Ambion) and preserved at 70°C before mRNA extraction, which used a commercially available assay (RNeasy, Qiagen). Medulla and cortex were separated and individual RNA was obtained for each of them. No pooling was performed. After extraction of RNA, cRNA was prepared and arrays performed using Agilent Human miRNA Microarrays (V3, release 12.0) performed at the Ramaciotti Gene Function Analysis facility, University of New South Wales in Sydney, Australia.
Project description:Seventh generation Exiqon locked nucleic acid miRCURY LNA microarrays were used to profile the expression of microRNAs in whole blood of patients with lung cancer and of clinically relevant controls without the disease (individuals with non-cancerous lung nodule, at high risk for cancer because of heavy smoking, or with lung cancer treated by surgical resection). The goal was to examine if whole blood microRNAs can be used for developing a non-invasive assay to diagnose lung cancer. Using the PAXgene Blood miRNA kit (Qiagen, Valencia, CA, USA), total RNA was isolated from whole blood stabilized and stored in PAXgene Blood RNA tubes for 85 individuals with lung cancer, 59 individuals without the disease but at a high risk for the disease because of smoking, and 17 individuals with a non-cancerous pulmonary nodule. RNA was also isolated from 13 samples of blood of 12 of the lung cancer cases following surgery; blood samples were collected on two different days post-operatively for one of the cases (Sample names 157 and 160). The data-set here includes data (sample name 171) from a post-operative blood sample of an individual (who had the lung cancer removed by operation) for whom good array data was not obtained for RNA from a pre-operative blood sample. Two RNA samples were used to test technical duplicability of the microarray platform (pairs of sample names 59 & 192, and 175 & 182). Thus, a total of 162 unique individuals and 175 unique RNA samples are covered by this data-set. Isolated RNA was provided to Exiqon (Denmark) for microRNA quantification using microarrays. Quality and concentration of RNA in the samples was assessed by Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA) and Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) instruments. Sample RNA (500 ng) was labeled with the Cy3-like Hy3 dye, mixed with 500 ng of a human universal reference RNA (product number AM6000, Ambion, Austin, TX, USA) that was labeled with the Cy5-like Hy5 dye, and hybridized to 7th generation miRCURY locked nucleic acid oligonucleotide microarrays. Sixty-two artificial small RNAs were spiked-in each RNA sample before labeling. disease state: Lung cancer (before surgical resection of lung cancer): 1, 2, 4, 5, 6, 7, 8, 9, 10, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 61, 63, 64, 65, 67, 68, 69, 70, 81, 82, 85, 86, 87, 88, 89, 90, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 121, 122, 123, 124, 125, 126, 127, 149, 151, 153, 154, 156, 158, 165, 167, 174, 175, 176, 177, 179, 180, 181, 183, 184, 186, 187, 188, 189, 193 disease state: Non-cancerous lung nodule: 128, 129, 130, 131, 133, 134, 135, 136, 137, 140, 141, 142, 144, 145, 146, 148, 182, 190 disease state: None (after surgical resection of lung cancer): 155, 157, 159, 160, 161, 162, 163, 164, 166, 169, 170, 171, 172, 173 disease state: None (no current or past lung cancer or non-cancerous nodule): 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 113, 114, 115, 116, 117, 118, 119, 120, 192 individual: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162
Project description:Analysis of four lung cancer cell lines transfected with a vector expressing the transcriptional repressor Snail versus a vector control. Aberrant Snail expression is known to induce an EMT program in lung cancers. Four lung cancer cell lines (H292, H358, H441, H1437) with stable overexpression of the human SNAI1 gene and vector expressing controls were collected at equal confluency with the miRNeasy Mini kit (Qiagen). One microgram of total RNA was labeled using miRCURY LNA™ microRNA Array Power Labeling kit by the UCLA Clinical Microarray Core. The labeled miRNAs were hybridized to Exiqon miRCURY LNA microRNA Array-6th Generation according to the manufacturer’s instructions. This array includes 927/648/351 human/mouse/rat miRNAs as well as 438 miRPlus miRNAs. The miRNA array slides were scanned using Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA) and processed by using the GenePix Pro 6.0 software (Axon Instruments). The raw data were normalized by using a combination of housekeeping miRNA, spike-in miRNA and invariant endogenous miRNAs.
Project description:To get to know the different levels of the microRNA among the pulmonary tuberculosis patients, latent-infected individuals and the complete healthy people, we have employed the Agilent miRNA microarray (version 2.0) as a discovery platform to identify microRNA with the potential to diagnose the TB patients and the latent infected persons. The Peripheral Blood Mononuclear Cells (PBMCs) of each group were made up of equally mixed quantity of 3 volunteers. The results showed that several microRNAs were found up- or low regulated in PBMCs of TB patients versus latent-infected individuals and similarly for latent-infected individuals versus complete healthy ones. Each group have 4 volunteers. The PBMCs of each group were made up of equally mixed volumes after being purified.each group was reated three times.
Project description:To determine the differential expression of miRNAs in the lungs of mice subjected to a model of allergic airways disease and non-allergic and steroid-treated control animals. Total lung RNA was collected from mice sensitised and challenged with PBS or OVA with or without DEX treatment at day 16 and miRNA microarrays performed.