High-Throughput Sequencing of Plasma MicroRNA in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Background

MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME.

Results

Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients.

Conclusion

Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.     

De-Regulated MicroRNAs in Pediatric Cancer Stem Cells Target Pathways Involved in Cell Proliferation,Cell Cycle and Development

Background

microRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.

Methodology/Results

Using a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).

Conclusion

Our findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.     

MicroRNA expression signatures of bladder cancer revealed by deep sequencing

Background

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They are aberrantly expressed in many types of cancers. In this study, we determined the genome-wide miRNA profiles in bladder urothelial carcinoma by deep sequencing.

Methodology/Principal Findings

We detected 656 differentially expressed known human miRNAs and miRNA antisense sequences (miRNA*s) in nine bladder urothelial carcinoma patients by deep sequencing. Many miRNAs and miRNA*s were significantly upregulated or downregulated in bladder urothelial carcinoma compared to matched histologically normal urothelium. hsa-miR-96 was the most significantly upregulated miRNA and hsa-miR-490-5p was the most significantly downregulated one. Upregulated miRNAs were more common than downregulated ones. The hsa-miR-183, hsa-miR-200b∼429, hsa-miR-200c∼141 and hsa-miR-17∼92 clusters were significantly upregulated. The hsa-miR-143∼145 cluster was significantly downregulated. hsa-miR-182, hsa-miR-183, hsa-miR-200a, hsa-miR-143 and hsa-miR-195 were evaluated by Real-Time qPCR in a total of fifty-one bladder urothelial carcinoma patients. They were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium (p<0.001 for each miRNA).

Conclusions/Significance

To date, this is the first study to determine genome-wide miRNA expression patterns in human bladder urothelial carcinoma by deep sequencing. We found that a collection of miRNAs were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium, suggesting that they might play roles as oncogenes or tumor suppressors in the development and/or progression of this cancer. Our data provide novel insights into cancer biology.     

Site-Specific MicroRNA Expression May Lead to Different Subtypes in Ulcerative Colitis

Background and Aim

Ulcerative Colitis (UC) is a type of inflammatory bowel disease, considered as an important disease of gastrointestinal tract having a huge impact on the health of the patient. Prolonged inflammation of colon in UC patients increases the risk of developing colorectal cancer. MiRNA are reported as a connecting link between inflammation and cancer. Differential miRNA expression is reported in Crohn’s disease (CD) patients involving various regions of the gastrointestinal tract. The current study was performed to dissect out the site specific miRNA expression in the colon biopsy samples of UC patients from Northern India.

Methods

Biopsy samples were collected from UC patients and healthy controls from Rectosigmoid Area (RS) and Ascending Colon (AC). MiRNA expression was compared between patients with RS and AC using a microarray platform. Differential expression was further validated by Real Time PCR analysis. Demographic and pathological data of UC -associated CRC patients was collected from the hospital database and analyzed for assessing the site of cancer.

Results

Upon analysis of data generated on a microarray platform and qRT PCR revealed that the expression of six miRNAs hsa-miR-146b-5p, hsa-miR-335-3p, hsa-miR-342-3p, hsa-miR-644b-3p, hsa-miR-491-3p, hsa-miR-4732-3p were downregulated in patients where RS was involved as compared to AC. The expression of hsa-miR-141-3p was upregulated in patients where RS region was involved as compared to AC. Analysis of the registered UC patient’s database from the hospital revealed that the site of CRC was predomimnantly the rectosigmoid region of the colon in most of the cases.

Conclusion

This is the first study to show the differential expression of miRNA involving different sites of colon in UC patients. Taking our data and previous reports into consideration, we propose that differential miRNA expression during UC perhaps contribute in the development of UC-associated CRC at the rectosigmoid area.     

A blood based 12-miRNA signature of Alzheimer disease patients

Background

Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples.

Results

We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies.

Conclusions

The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.     

Estimating survival time of patients with glioblastoma multiforme and characterization of the identified microRNA signatures

Background

Though glioblastoma multiforme (GBM) is the most frequently occurring brain malignancy in adults, clinical treatment still faces challenges due to poor prognoses and tumor relapses. Recently, microRNAs (miRNAs) have been extensively used with the aim of developing accurate molecular therapies, because of their emerging role in the regulation of cancer-related genes. This work aims to identify the miRNA signatures related to survival of GBM patients for developing molecular therapies.

Results

This work proposes a support vector regression (SVR)-based estimator, called SVR-GBM, to estimate the survival time in patients with GBM using their miRNA expression profiles. SVR-GBM identified 24 out of 470 miRNAs that were significantly associated with survival of GBM patients. SVR-GBM had a mean absolute error of 0.63 years and a correlation coefficient of 0.76 between the real and predicted survival time. The 10 top-ranked miRNAs according to prediction contribution are as follows: hsa-miR-222, hsa-miR-345, hsa-miR-587, hsa-miR-526a, hsa-miR-335, hsa-miR-122, hsa-miR-24, hsa-miR-433, hsa-miR-574 and hsa-miR-320. Biological analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the identified miRNAs revealed their influence in GBM cancer.

Conclusion

The proposed SVR-GBM using an optimal feature selection algorithm and an optimized SVR to identify the 24 miRNA signatures associated with survival of GBM patients. These miRNA signatures are helpful to uncover the individual role of miRNAs in GBM prognosis and develop miRNA-based therapies.

     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

Alterations in Circulating miRNA Levels following Early-Stage Estrogen Receptor-Positive Breast Cancer Resection in Post-Menopausal Women

Introduction

Circulating microRNAs (miRNAs) exhibit remarkable stability and may serve as biomarkers in several clinical cancer settings. The aim of this study was to investigate changes in the levels of specific circulating miRNA following breast cancer surgery and evaluate whether these alterations were also observed in an independent data set.

Methods

Global miRNA analysis was performed on prospectively collected serum samples from 24 post-menopausal women with estrogen receptor-positive early-stage breast cancer before surgery and 3 weeks after tumor resection using global LNA-based quantitative real-time PCR (qPCR).

Results

Numbers of specific miRNAs detected in the samples ranged from 142 to 161, with 107 miRNAs detectable in all samples. After correction for multiple comparisons, 3 circulating miRNAs (miR-338-3p, miR-223 and miR-148a) exhibited significantly lower, and 1 miRNA (miR-107) higher levels in post-operative vs. pre-operative samples (p<0.05). No miRNAs were consistently undetectable in the post-operative samples compared to the pre-operative samples. Subsequently, our findings were compared to a dataset from a comparable patient population analyzed using similar study design and the same qPCR profiling platform, resulting in limited agreement.

Conclusions

A panel of 4 circulating miRNAs exhibited significantly altered levels following radical resection of primary ER+ breast cancers in post-menopausal women. These specific miRNAs may be involved in tumorigenesis and could potentially be used to monitor whether all cancer cells have been removed at surgery and/or, subsequently, whether the patients develop recurrence.     

MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus

Background

Dysregulation of microRNA (miRNA) expression in various tissues and body fluids has been demonstrated to be associated with several diseases, including Type 2 Diabetes mellitus (T2D). Here, we compare miRNA expression profiles in different tissues (pancreas, liver, adipose and skeletal muscle) as well as in blood samples from T2D rat model and highlight the potential of circulating miRNAs as biomarkers of T2D. In parallel, we have examined the expression profiles of miRNAs in blood samples from Impaired Fasting Glucose (IFG) and T2D male patients.

Methodology/Principal Findings

Employing miRNA microarray and stem-loop real-time RT-PCR, we identify four novel miRNAs, miR-144, miR-146a, miR-150 and miR-182 in addition to four previously reported diabetes-related miRNAs, miR-192, miR-29a, miR-30d and miR-320a, as potential signature miRNAs that distinguished IFG and T2D. Of these microRNAs, miR-144 that promotes erythropoiesis has been found to be highly up-regulated. Increased circulating level of miR-144 has been found to correlate with down-regulation of its predicted target, insulin receptor substrate 1 (IRS1) at both mRNA and protein levels. We could also experimentally demonstrate that IRS1 is indeed the target of miR-144.

Conclusion

We demonstrate that peripheral blood microRNAs can be developed as unique biomarkers that are reflective and predictive of metabolic health and disorder. We have also identified signature miRNAs which could possibly explain the pathogenesis of T2D and the significance of miR-144 in insulin signaling.     

Discovery and Validation of a Prostate Cancer Genomic Classifier that Predicts Early Metastasis Following Radical Prostatectomy

Purpose

Clinicopathologic features and biochemical recurrence are sensitive, but not specific, predictors of metastatic disease and lethal prostate cancer. We hypothesize that a genomic expression signature detected in the primary tumor represents true biological potential of aggressive disease and provides improved prediction of early prostate cancer metastasis.

Methods

A nested case-control design was used to select 639 patients from the Mayo Clinic tumor registry who underwent radical prostatectomy between 1987 and 2001. A genomic classifier (GC) was developed by modeling differential RNA expression using 1.4 million feature high-density expression arrays of men enriched for rising PSA after prostatectomy, including 213 who experienced early clinical metastasis after biochemical recurrence. A training set was used to develop a random forest classifier of 22 markers to predict for cases - men with early clinical metastasis after rising PSA. Performance of GC was compared to prognostic factors such as Gleason score and previous gene expression signatures in a withheld validation set.

Results

Expression profiles were generated from 545 unique patient samples, with median follow-up of 16.9 years. GC achieved an area under the receiver operating characteristic curve of 0.75 (0.67–0.83) in validation, outperforming clinical variables and gene signatures. GC was the only significant prognostic factor in multivariable analyses. Within Gleason score groups, cases with high GC scores experienced earlier death from prostate cancer and reduced overall survival. The markers in the classifier were found to be associated with a number of key biological processes in prostate cancer metastatic disease progression.

Conclusion

A genomic classifier was developed and validated in a large patient cohort enriched with prostate cancer metastasis patients and a rising PSA that went on to experience metastatic disease. This early metastasis prediction model based on genomic expression in the primary tumor may be useful for identification of aggressive prostate cancer.     

Cell-Free MicroRNA Expression Profiles in Malignant Effusion Associated with Patient Survival in Non-Small Cell Lung Cancer

Objective

MicroRNAs (miRNAs) expression is altered in cancer cells, and miRNAs could serve as diagnostic and prognostic biomarker for cancer patients. This study was designed to analyze circulating miRNAs expression in the malignant pleural effusion (MPE) and their association with patient survival in non-small cell lung cancer (NSCLC).

Methods

Pleural effusion from 184 patients with NSCLC and MPE were collected. MiRNA microarray and bioinformatics interpretation were used to evaluate miRNA expression profiles in 10 NSCLC patients with different survival prognosis. Associations were validated in 184 patients (randomly classified into training and validation set with equal number in each group) using quantitative RT-PCR. Risk scores were formulated based on the expression signature of miRNAs. Clinical data, such as patient survival, were collected for correlation analysis.

Results

Thirty-three miRNAs were found to be altered more than two-fold by microarray in malignant effusions between longer-survival and shorter-survival groups, and levels of five miRNAs (miRNA-93, miRNA-100, miRNA-134, miRNA-151 and miRNA-345) were significantly associated with overall survival. High expression of miR-100 and low expression of miRNA-93, miRNA-134, miRNA-151 and miRNA-345 were associated with poor survival in both the training and validation cohort. Patients with high risk scores had overall poor survival compared to the patients with low risk scores. Risk score was an independent predictor of patient survival.

Conclusions

Expression patterns of miRNAs are systematically altered in MPE of patient with NSCLC. The five miRNA signature from the effusion may serve as a predictor for the overall survival of patients with lung cancers.     

Disease-related changes in the cerebrospinal fluid metabolome in amyotrophic lateral sclerosis detected by GC/TOFMS

Background/Aim

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.

Methodology

Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.

Principal Findings

The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.

Conclusions/Significance

Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.     

Reorganization of metastamiRs in the evolution of metastatic aggressive neuroblastoma cells

Background

MetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes.

Results

Whole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs’ targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKβ, Birc1, Birc2, Bric5, β-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3β, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1β, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs’ targets corresponded strongly with poor overall and relapse-free survival.

Conclusions

For the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1642-x) contains supplementary material, which is available to authorized users.