miRNA Fingerprints Research Articles

Circulating microRNA Signatures in Rodent Models of Pain.

MicroRNAs (miRNAs) remain stable in circulation and have been identified as potential biomarkers for a variety of conditions. We report miRNA changes in blood from multiple rodent models of pain, including spinal nerve ligation and spared nerve injury models of neuropathic pain; a complete Freund's adjuvant (CFA) model of inflammatory pain; and a chemotherapy-induced model of pain using the histone deacetylase inhibitor JNJ-26481585. The effect of celecoxib, a cyclooxygenase-2-selective nonsteroidal anti-inflammatory drug, was investigated in the CFA model as proof of principle for assessing the utility of circulating miRNAs as biomarkers in determining treatment response. Each study resulted in a unique miRNA expression profile. Despite differences in miRNAs identified from various models, computational target prediction and functional enrichment have identified biological pathways common among different models. The Wnt signaling pathway was affected in all models, suggesting a crucial role for this pathway in the pathogenesis of pain. Our studies demonstrate the utility of circulating miRNAs as pain biomarkers and suggest the potential for rigorous forward and reversetranslational approaches. Evaluating alterations in miRNA fingerprints under different pain conditions and after administering therapeutic agents may be beneficial in evaluating clinical trial outcomes, predicting treatment response, and developing correlational outcomes between preclinical and human studies.

Serum microRNAs in sporadic amyotrophic lateral sclerosis

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and specific mircoRNA “fingerprints” are thought to contribute to and/or reflect certain disease conditions. Recently, we identified surprisingly homogeneous signatures of circulating miRNAs in the serum of familial amyotrophic lateral sclerosis (ALS) patients, which were already present in presymptomatic carriers of ALS gene mutations. Here, we characterize circulating miRNAs in the serum of sporadic ALS patients. We show that, in contrast to familial ALS, miRNA signatures of sporadic ALS are highly heterogeneous suggesting a number of different etiologies. Nevertheless, 2 miRNAs, miR-1234-3p and miR-1825, could be identified to be consistently downregulated in sporadic ALS. Bioinformatic analysis revealed miRNA fingerprints resembling those of familial ALS patients and mutation carriers in 61% of sporadic ALS patients, while the remaining subgroup had clearly different miRNA signatures. These data support a higher than expected contribution of genetic factors also to sporadic ALS. Moreover, our results indicate a more heterogeneous molecular etiology of sporadic ALS compared with (mono)genic cases, which should be considered for the development of disease modifying treatments.

Differentially regulated miRNAs as prognostic biomarkers in the blood of primary CNS lymphoma patients

Despite improved therapeutic regimens, primary CNS lymphoma (PCNSL) remains a therapeutic challenge. A prognostic classification of PCNSL patients may represent an important step towards optimised patient-adapted therapy. However, only higher age and low Karnofsky Performance Status (KPS) have repeatedly been reported to be associated with shorter overall survival (OS).Here we characterised microRNA (miRNA) fingerprints in the blood of PCNSL patients with short-term survival (STS) versus long-term survival (LTS) to assess their potential as novel prognostic biomarkers. Blood was collected from patients enrolled in the G-PCNSL-SG1 trial, a phase III study for patients with newly diagnosed PCNSL. miRNAs were extracted from the blood and analysed by next generation sequencing.The STS group comprised 20 patients with a median OS of 3months and was compared to 20 LTS patients with a median OS of 55months. The cohorts were balanced for age and KPS. Twelve annotated miRNAs were significantly deregulated between the two groups. Among them, miR-151a-5p and miR-151b exhibited the most prominent differences. Importantly, the combination of several miRNA allowed for a good separation between short- and long-term survivors with maximal Area Under Curve (AUC) above 0.75. Besides the known miRNAs we identified putative novel miRNA candidates with potential regulatory influence of PCNSL. Finally, the differential regulation of the most promising candidate miRNAs was confirmed by real-time polymerase chain reaction (PCR) in a validation cohort consisting of 20 STS and LTS patients.In conclusion, peripheral blood miRNA expression patterns hold promise as a prognostic tool in PCNSL patients.

MicroRNA Profiling Reveals Unique miRNA Signatures in IGF-1 Treated Embryonic Striatal Stem Cell Fate Decisions in Striatal NeurogenesisIn Vitro

The striatum is considered to be the central processing unit of the basal ganglia in locomotor activity and cognitive function of the brain. IGF-1 could act as a control switch for the long-term proliferation and survival of EGF + bFGF-responsive cultured embryonic striatal stem cell (ESSC), while LIF imposes a negative impact on cell proliferation. The IGF-1-treated ESSCs also showed elevated hTERT expression with demonstration of self-renewal and trilineage commitment (astrocytes, oligodendrocytes, and neurons). In order to decipher the underlying regulatory microRNA (miRNA)s in IGF-1/LIF-treated ESSC-derived neurogenesis, we performed in-depth miRNA profiling at 12 days in vitro and analyzed the candidates using the Partek Genome Suite software. The annotated miRNA fingerprints delineated the differential expressions of miR-143, miR-433, and miR-503 specific to IGF-1 treatment. Similarly, the LIF-treated ESSCs demonstrated specific expression of miR-326, miR-181, and miR-22, as they were nonsignificant in IGF-treated ESSCs. To elucidate the possible downstream pathways, we performed in silico mapping of the said miRNAs into ingenuity pathway analysis. Our findings revealed the important mRNA targets of the miRNAs and suggested specific interactomes. The above studies introduced a new genre of miRNAs for ESSC-based neuroregenerative therapeutic applications.

Signature of Circulating MicroRNAs as Potential Biomarkers in Vulnerable Coronary Artery Disease

AimsMicroRNAs (miRNAs) play important roles in the pathogenesis of cardiovascular diseases. Circulating miRNAs were recently identified as biomarkers for various physiological and pathological conditions. In this study, we aimed to identify the circulating miRNA fingerprint of vulnerable coronary artery disease (CAD) and explore its potential as a novel biomarker for this disease.Methods and ResultsThe Taqman low-density miRNA array and coexpression network analyses were used to identify distinct miRNA expression profiles in the plasma of patients with typical unstable angina (UA) and angiographically documented CAD (UA group, n = 13) compared to individuals with non-cardiac chest pain (control group, n = 13). Significantly elevated expression levels of miR-106b/25 cluster, miR-17/92a cluster, miR-21/590-5p family, miR-126*, and miR-451 were observed in UA patients compared to controls. These findings were validated by real-time PCR in another 45 UA patients, 31 stable angina patients, and 37 controls. In addition, miR-106b, miR-25, miR-92a, miR-21, miR-590-5p, miR-126* and miR-451 were upregulated in microparticles (MPs) isolated from the plasma of UA patients (n = 5) compared to controls (n = 5). Using flow cytometry and immunolabeling, we further found that Annexin V+ MPs were increased in the plasma samples of UA patients compared to controls, and the majority of the increased MPs in plasma were shown to be Annexin V+ CD31+ MPs. The findings suggest that Annexin V+ CD31+ MPs may contribute to the elevated expression of the selected miRNAs in the circulation of patients with vulnerable CAD.ConclusionThe circulating miRNA signature, consisting of the miR-106b/25 cluster, miR-17/92a cluster, miR-21/590-5p family, miR-126* and miR-451, may be used as a novel biomarker for vulnerable CAD.Trial RegistrationChinese Clinical Trial Register, ChiCTR-OCH-12002349.

Improvements to Direct Quantitative Analysis of Multiple MicroRNAs Facilitating Faster Analysis

Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.

Identification of microRNAs in blood and urine as tumour markers for the detection of urinary bladder cancer

Since differential expression of microRNAs (miRNAs) has been found to be highly associated with several types of cancer, the goal of the present study was to identify an miRNA fingerprint as a non‑invasive diagnostic tool to detect urinary bladder cancer using the easily accessible samples of whole blood and urine. Blood and urine samples from 4 controls and from patients suffering from superficial and invasive bladder cancer were analyzed using miRNA microarray consisting of 754 human miRNAs from the Sanger database v14. Using RT‑qPCR technique, 6 of the differentially expressed miRNAs were validated in the controls (20 blood, 19 urine samples) and patients with superficial (18 blood, 16 urine samples) or invasive (20 blood and urine samples each) tumours. Three blood miRNAs (miR‑26b‑5p, miR‑144‑5p, miR‑374‑5p) were found to be significantly upregulated in invasive bladder tumour patients (P<0.05) when compared to the control group. The expression of 2 miRNAs (miR‑618, miR‑1255b‑5p) in the urine of patients with invasive tumours was significantly (P<0.05) increased in comparison to the control group. Blood miR‑26b‑5p detected the presence of invasive bladder tumours with 94% specificity and 65% sensitivity. The urine miR‑1255b‑5p reached 68% specificity and 85% sensitivity in the diagnosis of invasive tumours. This pilot study represents the first characterization of an miRNA profile for urinary bladder tumours in whole blood samples. In addition, it was shown that invasive bladder tumours could be identified by differentially expressed urine miRNAs. Further studies are needed to test the clinical usefulness for bladder cancer detection and surveillance.

Cell‐free microRNAs as diagnostic, prognostic, and predictive biomarkers for lung cancer

Lung cancer is the most common cancer worldwide, accounting for over 1.37 million deaths annually. The clinical outcome and management of lung cancer patients could be substantially improved by the implementation of non-invasive biomarker assays for the early detection, prognosis as well as prediction and monitoring of treatment response. MicroRNAs (miRNAs) have been implicated in the regulation of virtually all signaling circuits within a cell and their dysregulation has been shown to play an essential role in the development and progression of cancer. Recently, miRNAs were found to be released into the circulation and to exist there in a remarkably stable form. Furthermore, various cancers were shown to leave specific miRNA fingerprints in the blood of patients suggesting that cell-free miRNAs could serve as non-invasive biomarkers for the detection or monitoring of cancer and putative therapeutic targets. Since that, a considerable effort has been devoted to decode the information carried by circulating miRNAs. In the current review, we give an insight into the mechanisms of miRNA release into the bloodstream, their putative functional significance and systematically review the studies focused on the identification of cell-free miRNAs with the diagnostic, prognostic, and predictive significance in lung cancer and discuss their potential clinical utility.

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms

AbstractPosttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

Identification of circulating microRNAs as biomarkers in cancers: what have we got?

During the past few years there has been great interest in the development of circulating microRNAs (miRNAs) as stable blood-based biomarkers for cancer detection. Deregulation of miRNAs in blood samples has shown considerable clinical utilities in cancers. Due to poorly characterized preanalytical and analytical variables and the lack of a standardized measurement protocol, the application of these miRNA fingerprints is hindered by conflicting results. In this review,we outline our current understanding of preanalytically and analytically confounding factors. We believe that great consideration should be taken in the development of circulating miRNA as tumor biomarkers.

149P - Microrna Profiling and CDDP-GPG DNA Adduct Formation Analysis in a Panel of Cisplatin Resistant Non-Small Cell Lung Cancer Cell Lines

ABSTRACT Introduction The outcome of cisplatin therapy in NSCLC has reached a plateau, with the development of resistance being a major obstacle in the use of this drug. Understanding the molecular mechanisms underlying this resistance phenotype may aid in the development of novel agents that enhance the sensitivity of cisplatin. In this study, we examined the microRNA profile and levels of cisplatin-DNA adduct formation in a panel of cisplatin resistant NSCLC cell lines. Methods We have previously generated a panel of cisplatin resistant (CisR) cell lines (MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells and extensively characterised these based on a number of functional assays including proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability (clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by FACS and Western blot analysis. MicroRNA profiling was carried out using the nCounter miRNA Expression Assay consisting of 749 target miRNA's. Immunofluorescence staining and measurement of specific DNA platination products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using immunofluorescence microscopy and quantitative digital image analysis using the ACAS 6.0 CytometryAnalysis System, respectively. Results In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype, a 3- and 13-miRNA signature was deduced, based on differential expression of 3 miRNA's (5/5 cell lines) and 13 miRNA's (4/5 cell lines) in the CisR cell lines relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly reduced in resistant cells up to 24h in response to cisplatin compared to that observed in corresponding parent cells. Conclusion We have identified a distinct miRNA fingerprint in a clinically relevant, isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in vitro, these may be used as a potential predictive marker in patient response to platinum therapy in NSCLC. Disclosure All authors have declared no conflicts of interest.

MicroRNA expression profiles of trophoblastic cells

BackgroundMicroRNAs (miRNAs) are small single-stranded RNA molecules working as post-transcriptional modulators of gene expression. Trophoblast cells are a heterogenous group of fetal cells forming the feto–maternal interface and displaying a wide spectrum of functions. The regulation of their behavior may partly underly the control through miRNAs. Therefore, we aimed to compare the miRNA profile of primary first and third trimester trophoblast cells with that of different trophoblastic cell lines. Material and methodsTotal RNA was obtained from isolated cytotrophoblast cells from healthy term and first trimester placentae and the cell lines HTR-8/SVneo (immortalized trophoblast cells), JEG-3 (choriocarcinoma), ACH-3P and AC1-M59, which are choriocarcinoma cells fused with first and third trimester trophoblast cells, respectively. The expression level of 762 different miRNAs was quantitatively analyzed by using a TaqMan Human MicroRNA Array. For testing the reproducibility of the array technique, the expression of 9 selected miRNAs has been re-analyzed by individual qPCR. ResultsThe analyzed cell types share many similar patterns of miRNAs, but are significantly distinct in the expression of three miRNA clusters: chromosome 19 miRNA cluster (C19MC; containing 54 different miRNAs), C14MC (34 miRNAs) and a minor cluster (miRNA-371 to miRNA-373 cluster), also located on chromosome 19. Expression of miRNAs within C19MC increases significantly from first to third trimester trophoblast while that of C14MC members decreases. MiRNAs within the miR-371-3 cluster augment slightly. C19MC and the miR-371-3 cluster are not expressed by HTR-8/SVneo cells whilst C14MC is almost not detectable in the choriocarcinoma-derived cell lines complete array data available at NCBI Gene Expression Omnibus accession number GSE32346: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32346). Beside the miRNAs within the mentioned clusters, further 27 miRNAs are differentially expressed (>100 fold) between term and first trimester trophoblast cells. The placenta-specific miRNAs miR-141 and miR-21 as well as let-7g are expressed in all tested cells with the highest expression in primary trophoblast cells. ConclusionPrimary first trimester and term trophoblast cells and trophoblastic cell lines display major differences in their miRNA fingerprints which may be involved in their different behavior and characteristics.

Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection

MicroRNAs (miRNAs) repress the expression levels of genes by binding to mRNA transcripts, acting as master regulators of cellular processes. Differential expression of miRNAs has been linked to virus-associated diseases involving members of the Hepacivirus, Herpesvirus, and Retrovirus families. In contrast, limited biological and molecular information has been reported on the potential role of cellular miRNAs in the life cycle of influenza A viruses (infA). In this study, we hypothesize that elucidating the miRNA expression signatures induced by low-pathogenicity swine-origin infA (S-OIV) pandemic H1N1 (2009) and highly pathogenic avian-origin infA (A-OIV) H7N7 (2003) infections could reveal temporal and strain-specific miRNA fingerprints during the viral life cycle, shedding important insights into the potential role of cellular miRNAs in host-infA interactions. Using a microfluidic microarray platform, we profiled cellular miRNA expression in human A549 cells infected with S- and A-OIVs at multiple time points during the viral life cycle, including global gene expression profiling during S-OIV infection. Using target prediction and pathway enrichment analyses, we identified the key cellular pathways associated with the differentially expressed miRNAs and predicted mRNA targets during infA infection, including the immune system, cell proliferation, apoptosis, cell cycle, and DNA replication and repair. By identifying the specific and dynamic molecular phenotypic changes (microRNAome) triggered by S- and A-OIV infection in human cells, we provide experimental evidence demonstrating a series of temporal and strain-specific host molecular responses involving different combinatorial contributions of multiple cellular miRNAs. Our results also identify novel potential exosomal miRNA biomarkers associated with pandemic S-OIV and deadly A-OIV-host infection.

A specific miRNA signature in the peripheral blood of glioblastoma patients

The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood-derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age- and sex-matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student's t-test at an alpha level of 0.05. Of these, two candidates, miR-128 (up-regulated) and miR-342-3p (down-regulated), remained significant after correcting for multiple testing by Benjamini-Hochberg adjustment with a p-value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real-time PCR and validated for patients who had received neither radio- nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78-84%], specificity of 79% (95% CI 75-83%) and sensitivity of 83% (95% CI 71-85%). In summary, our proof-of-concept study demonstrates that blood-derived glioblastoma-associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.

Abstract 4945: Androgen-mediated phenotypic change produces a miRNA fingerprint in prostate carcinoma cells

Abstract Activation of the androgen receptor (AR) results in phenotypic change in androgen-responsive cells; for example, in the human prostate carcinoma cell line (LNCaP) androgens mediate cell proliferation. Factors controlling the range of phenotypic responses mediated by androgens are only partially understood, but include both transcriptional and post-transcriptional mechanisms. One such mechanism that appears to offer wide ranging, yet highly selective control, is microRNA-mediated post-transcriptional regulation. In the present study, we assessed the influence of endocrine-active compounds that act via the AR pathway on the microRNA (miRNA) profile. As negative regulators of gene expression, miRNA are short non-coding RNA that are able to suppress translation or induce degradation of their target mRNA transcripts leading to decreased protein expression. The effect of exposure of LNCaP cells to a model synthetic AR agonist, a physiological androgen and a therapeutic AR antagonist was determined at various time points up to 120 hours. Phenotypic response to exposure was assessed as an alteration of proliferative ability, expression of prostate-specific antigen (PSA) and alteration of the miRNA-ome. As expected, the agonists significantly induced proliferation and PSA expression, compared to vehicle control, whilst the antagonist itself had no effect on proliferation and PSA expression except when cultured in the presence of the natural ligand resulting in potentiation of the ligand's effects. The temporal effects of the compounds on the expression profile of 866 miRNAs were assessed using Agilent's miRNA microarray version 3. A number of miRNAs were found to be altered in a compound-specific and temporal manner, including some implicated in cell cycle control. qRT-PCR of select miRNAs proceeded to validate the fold changes observed by microarray. Targets for altered miRNAs were deduced using bio-informatic approaches and immunoblotting of target proteins has confirmed that protein levels are inversely correlated. These data describe an androgen-mediated miRNA ‘fingerprint’ that is compound and temporally dependent and provides mechanistic insights into the biological responses of cells to such compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4945. doi:10.1158/1538-7445.AM2011-4945

Role of microRNAs in drug-resistant ovarian cancer cells

Objectives Chemotherapy is the preferred therapeutic approach for the therapy of advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This work was aimed to identify selected microRNAs involved in the development of chemoresistance in ovarian cancer. Methods High-throughput analysis of the miRNA profile in a panel of paclitaxel- (A2780TAX, A2780TC1 and A2780TC3) and cisplatin-resistant (A2780CIS) cells was assessed using a microarray platform and subsequent validation with qPCR and Northern blots. Downstream target validation was performed for miR-130a and the target M-CSF.] Results Six miRNAs (let-7e, miR-30c, miR-125b, miR-130a and miR-335) were always diversely expressed in all the resistant cell lines. Let-7e was upregulated in A2780TAX cells, while it was downregulated in the other resistant cell lines. The opposite phenomenon was obtained for miR-125b, which was downregulated in A2780TAX and upregulated in the other cell lines. The miR-30c, miR-130a and miR-335 were downregulated in all the resistant cell lines, thereby suggesting a direct involvement in the development of chemoresistance. Finally downstream target validation was proven for the miR-130a, whose downregulation was linked to the translational activation of the M-CSF gene, a known resistance factor for ovarian cancer. Conclusions Our results indicate that ovarian cancer drug resistance is associated with a distinct miRNA fingerprint, and miRNA microarrays could represent a prognostic tool to monitor the chemotherapy outcome.