Identification of mouse serum miRNA endogenous references by global gene expression profiles.
ABSTRACT: MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.
Project description:MiRNAs of blood and urine have been shown to represent a convenient source of biomarkers for prostate cancer (PCa) diagnosis and assessment of the therapy effectiveness due to their high stability and representation and the low invasiveness of sample collection. Here, we studied the influence of radical prostatectomy (RP) on the expression of 12 cell-free miRNAs previously shown as potential markers of PCa (i.e., miR-19b, miR-22, miR-92a, miR-378, miR-425, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375 and miR-660). The relative expression of the miRNAs combined into 31 paired ratios was evaluated in the urine extracellular vesicles (EVs), clarified urine (CU) and blood plasma of healthy donors, pre- and post-RP samples of PCa patients. Nineteen miRNA ratios based on combinations of ten of the miRNAs (miR-19b, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375, miR-378, miR-425, and miR-660) were altered by RP. The comparative expression analysis of the cell-free miRNA ratios between healthy donors and PCa patients revealed miR-125b/miR-30e and miR-375/miR-30e as potential markers for evaluating therapeutic efficacy. MiR-378/miR-19b, miR-425/miR-19b, miR-200/miR-30e, miR-660/miR-30e, and miR-205/miR-30e had minor prognostic value but could be used to increase the steadiness of the diagnostic system. The urine EVs had the highest potential as a source of markers.
Project description:Prostate cancer is a global biological, medical, and social issue aggravated by the lack of reliable, highly specific, and sensitive non-invasive tests for diagnosis and staging of prostate cancer. One prospective source of biomarkers are the cell-free miRNAs present in various biological fluids. In the present study, we validated the diagnostic potential of cell-free miRNAs: miR-19b, miR-22, miR-92a, miR-378, miR-425, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375, and miR-660; we estimated the required sample size and the minimal miRNA set for a subsequent large-scale validation study. Relative expression of 12 miRNA combined in 31 ratios was investigated in three fractions of biological fluids (urine extracellular vesicles, clarified urine, and plasma) obtained from patients with prostate cancer (n = 10), benign prostate hyperplasia (n = 8), and healthy volunteers (n = 11). Eight of the miRNAs found in urine vesicles (miR-19b, miR-30e, miR-31, miR-92a, miR-125, miR-200, miR-205, and miR-660) showed great promise and when combined into six ratios (miR-125b/miR-30e, miR-200/miR-30e, miR-205/miR-30e, miR-31/miR-30e, miR-660/miR-30e, and miR-19b/miR-92a) could classify patients with prostate cancer, benign prostate hyperplasia, and healthy donors with 100% specificity, 100% sensitivity, and with a high degree of reliability for most donors.
Project description:The NONcNZO10/LtJ mouse is a polygenic model of type-2 diabetes (T2D) that shows moderate obesity and diabetes, and is regarded as a good model reflective of the conditions of human T2D. In this study, we analyzed pathological changes of pancreases with the progress of time by using histopathology and gene expression analysis, including microRNA. A number of gene expression changes associated with decreased insulin secretion (possibly regulated by miR-29a/b) were observed, and zinc homeostasis (Slc30a8, Mt1 and Mt2) or glucose metabolism (Slc2a2) was suggested as being the candidate mechanism of pancreas failure in NONcNZO10/LtJ mice. These results demonstrate NONcNZO10/LtJ mice have a complex pathogenic mechanism of diabetes, and moreover, this fundamental information of NONcNZO10/LtJ mice would offer the opportunity for research and development of a novel antidiabetic drug. To identify the diabetes related genes that changed expression during the development of T2D in NONcNZO10/LtJ mice, we analyzed gene expression profiles of 7-, 13-, and 22-week old NONcNZO10/LtJ mice using GeneChip Mouse Gene 2.0 ST Arrays. On the other hand, we compared gene expression profiles of 7-week old NONcNZO10/LtJ mice with 7-week old NON/ShiLtJ mice to investigate the differences of gene expression before developing T2D. Furthermore, to validate the hypothesis generated by the results of mRNA analysis, we identified differentially expressed microRNAs in 13- and 22-week compared with 7-week old NONcNZO10/LtJ mice using Affymetrix miRNA GeneChip 3.0 Arrays.
Project description:BACKGROUND: MicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas. METHODS: We measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis. RESULTS: Expression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu. CONCLUSIONS: Aberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.
Project description:BACKGROUND:The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach. RESULTS:We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p =?0.0002) and 2 miRNAs (p =?0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets. CONCLUSIONS:No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.
Project description:Objective:To determine a predictive factor for the risk of conversion from ocular myasthenia gravis (OMG) to generalized MG (GMG) in a prospective study. Methods:RNA was isolated from serum samples and detection of microRNA (miRNA) expression analyzed with qPCR. In the discovery set, 179 human miRNAs were assayed for profiling of five OMG patients and four age- and gender-matched healthy controls. Based on the specific accumulation pattern of 19 miRNAs from the discovery set, in addition to miRNAs previously found elevated in generalized MG (GMG; miR-150-5p and miR-30e-5p), 21 miRNAs were subsequently analyzed in a validation cohort of 83 OMG patients (82 immunosuppression treatment naive; 49 male) within 3 months of diagnosis and at a follow-up visit (median duration 28 months from first visit). Results:Thirteen patients generalized 14.8 ± 12.0 months after the diagnosis and the majority (85%) belonged to the late onset MG group. Two miRNAs were significantly higher in secondary GMG (SGMG) patients compared to OMG patients with late onset MG: miR-30e-5p (9.1 ± 0.5 vs. 6.3 ± 0.9; P < 0.0001) and miR-150-5p (7.4 ± 1.1 vs. 6.4 ± 1.1; P = 0.01). The sensitivity for miR-30e-5p in differentiating OMG and SGMG was 96% in all OMG patients and 100% in late onset OMG patients. Interpretation:This is the first study to describe a potential predictive factor associated with the risk of generalization for patients with OMG. Raised levels (>8) of miR-30e-5p at initial presentation in patients with ocular MG symptoms, give a predictive cut-off for subsequent generalization of 96-100%.
Project description:MicroRNA (miRNA) regulates the expression of genes and influences a series of biological processes, including fatty acid metabolism. We screened the expression of miRNA in goat mammary glands during peak-lactation and non-lactating ("dry") periods, and performed an in vitro study with goat mammary epithelial cells (GMEC) prior to sequencing analysis. Results illustrated that miR-30e-5p and miR-15a were highly expressed. Utilizing a luciferase reporter assay and Western blot, low-density lipoprotein receptor-related protein 6 (LRP6) and Yes associated protein 1 (YAP1) genes were demonstrated to be a target of miR-30e-5p and miR-15a in GMEC. Moreover, we demonstrated that the overexpression of miR-30e-5p and miR-15a in GMEC promoted fat metabolism while their knockdown impaired fat metabolism. These findings extend the discovery of a key role of miR-30e-5p and miR-15a in mediating adipocyte differentiation by suggesting a role in promoting milk fat synthesis. In conclusion, our findings indicate that miR-30e-5p, together with miR-15a, represses expression of LRP6 and promotes fat metabolism in GMEC. The data expanded our knowledge on the function of miRNAs in milk fat metabolism and synthesis in ruminant mammary cells.
Project description:BACKGROUND:Multi-gene expression assays are an attractive tool in revealing complex regulatory mechanisms in living organisms. Normalization is an indispensable step of data analysis in all those studies, since it removes unwanted, non-biological variability from data. In targeted qPCR assays it is typically performed with respect to prespecified reference genes, but the lack of robust strategy of their selection is reported in literature, especially in studies concerning circulating microRNAs (miRNA). Unfortunately, this problem impedes translation of scientific discoveries on miRNA biomarkers into widely available laboratory assays. Previous studies concluded that averaged expressions of multi-miRNA combinations are more stable references than single genes. However, due to the number of such combinations the computational load is considerable and may be hindering for objective reference selection in large datasets. Existing implementations of normalization algorithms (geNorm, NormFinder and BestKeeper) have poor performance and may require days to compute stability values for all potential reference as the evaluation is performed sequentially. RESULTS:We designed NormiRazor - an integrative tool which implements those methods in a parallel manner on a graphics processing unit (GPU) using CUDA platform. We tested our approach on publicly available miRNA expression datasets. As a result, the times of executions on 8 datasets containing from 50 to 400 miRNAs (subsets of GSE68314) decreased 18.7 ±0.6 (mean ±SD), 104.7 ±4.2 and 76.5 ±2.2 times for geNorm, BestKeeper and NormFinder with respect to previous Python implementation. To allow for easy access to normalization pipeline for biomedical researchers we implemented NormiRazor as an online platform where a user could normalize their datasets based on the automatically selected references. It is available at norm.btm.umed.pl, together with instruction manual and exemplary datasets. CONCLUSIONS:NormiRazor allows for an easy, informed choice of reference genes for qPCR transcriptomic studies. As such it can improve comparability and repeatability of experiments and in longer perspective help translate newly discovered biomarkers into readily available assays.
Project description:Malignant Pleural Mesothelioma (MPM) is an aggressive cancer mainly caused by asbestos exposure and refractory to current therapies. Specific diagnostic markers for early MPM diagnosis are needed. Changes in miRNA expression have been implicated in several diseases and cancers, including MPM. We examined if a specific miRNA signature in plasmatic extracellular vesicles (EV) may help to discriminate between malignant pleural mesothelioma patients (MPM) and subjects with Past Asbestos Exposure (PAE).We investigated 23 MPM patients and 19 cancer-free subjects with past asbestos exposure (PAE). We screened 754 miRNAs in plasmatic EVs by OpenArray and found 55 differential miRNAs using logistic regression models adjusted for age, sex, BMI, and smoking. Among the top-20 differential miRNAs chosen for validation by Real time PCR, 16 were confirmed. Using receiver operating characteristic (ROC) curve analysis, the most discriminating miRNA combination was given by miR-103a-3p + miR-30e-3p, which generated an AUC of 0.942 (95% CI 0.87-1.00), with a sensitivity of 95.5% and a specificity of 80.0%. Using multivariate Cox regression analysis including gender, age, BMI and smoking we found a Hazard Ratio for miR-103a-3p above the median of 0.37 (95%CI 0.13-1.13) and of 0.51 (95%CI 0.17-1.52) for miR-30e-3p.This study suggests EV-associated miR-103a-3p and miR-30e-3p are able to discriminate MPM from PAE subjects. Larger and prospective studies are needed to confirm these two-miRNA signature alone or in combination with other biomarkers as diagnostic tools for MPM.
Project description:Micro-ribonucleic acids (miRNAs) are small functional non-coding RNAs that downregulate gene expression at the post-transcriptional level. Abnormal expression of specific miRNAs has been recorded in chronic lymphocytic leukaemia, other non-Hodgkin B-cell lymphomas, lung cancer and chronic myeloid leukaemia (CML). The aim of this study was to compare miRNA expression profiles among patients with newly diagnosed CML, those on established therapy with imatinib mesylate, and healthy individuals. The expression of 88 miRNAs was evaluated in a total of nine samples divided into three groups: Group 1 comprised three samples collected from newly diagnosed CML patients; group 2 consisted of three samples collected from patients on therapy; the remaining three samples were collected from healthy volunteers (control group). Total RNA was extracted from whole blood and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed on the LightCycler® 480 platform using Human Serum & Plasma miRNA PCR Arrays. In group 1, only SNORD44 was downregulated, while hsa-miR-372 and hsa-miR-375 were found to be significantly upregulated compared with the control group. By contrast, 49 miRNAs were significantly upregulated in group 2 compared with the control group. miRNAs hsa-miR-106b, hsa-miR-21, hsa-miR-221, hsa-miR-10a, hsa-miR-193a-5p and hsa-miR-30e were expressed in group 2. Therefore, miRNA expression profiles differed between the two patient groups.