Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse.

The adaptive response to extreme endurance exercise might involve transcriptional and translational regulation by microRNAs (miRNAs). Therefore, the objective of the present study was to perform an integrated analysis of the blood transcriptome and miRNome (using microarrays) in the horse before and after a 160 km endurance competition. A total of 2,453 differentially expressed genes and 167 differentially expressed microRNAs were identified when comparing pre- and post-ride samples. We used a hypergeometric test and its generalization to gain a better understanding of the biological functions regulated by the differentially expressed microRNA. In particular, 44 differentially expressed microRNAs putatively regulated a total of 351 depleted differentially expressed genes involved variously in glucose metabolism, fatty acid oxidation, mitochondrion biogenesis, and immune response pathways. In an independent validation set of animals, graphical Gaussian models confirmed that miR-21-5p, miR-181b-5p and miR-505-5p are candidate regulatory molecules for the adaptation to endurance exercise in the horse. To the best of our knowledge, the present study is the first to provide a comprehensive, integrated overview of the microRNA-mRNA co-regulation networks that may have a key role in controlling post-transcriptomic regulation during endurance exercise.

MicroRNA Expression Array Identifies Novel Diagnostic Markers for Conventional and Oncocytic Follicular Thyroid Carcinomas

Objective:The most difficult thyroid tumors to be diagnosed by cytology and histology are conventional follicular carcinomas (cFTCs) and oncocytic follicular carcinomas (oFTCs). Several microRNAs (miRNAs) have been previously found to be consistently deregulated in papillary thyroid carcinomas; however, very limited information is available for cFTC and oFTC. The aim of this study was to explore miRNA deregulation and find candidate miRNA markers for follicular carcinomas that can be used diagnostically.Design:Thirty-eight follicular thyroid carcinomas (21 cFTCs, 17 oFTCs) and 10 normal thyroid tissue samples were studied for expression of 381 miRNAs using human microarray assays. Expression of deregulated miRNAs was confirmed by individual RT-PCR assays in all samples. In addition, 11 follicular adenomas, two hyperplastic nodules (HNs), and 19 fine-needle aspiration samples were studied for expression of novel miRNA markers detected in this study.Results:The unsupervised hierarchical clustering analysis demonstrated individual clusters for cFTC and oFTC, indicating the difference in miRNA expression between these tumor types. Both cFTCs and oFTCs showed an up-regulation of miR-182/-183/-221/-222/-125a-3p and a down-regulation of miR-542-5p/-574-3p/-455/-199a. Novel miRNA (miR-885-5p) was found to be strongly up-regulated (>40-fold) in oFTCs but not in cFTCs, follicular adenomas, and HNs. The classification and regression tree algorithm applied to fine-needle aspiration samples demonstrated that three dysregulated miRNAs (miR-885-5p/-221/-574-3p) allowed distinguishing follicular thyroid carcinomas from benign HNs with high accuracy.Conclusions:In this study we demonstrate that different histopathological types of follicular thyroid carcinomas have distinct miRNA expression profiles. MiR-885-5p is highly up-regulated in oncocytic follicular carcinomas and may serve as a diagnostic marker for these tumors. A small set of deregulated miRNAs allows for an accurate discrimination between follicular carcinomas and hyperplastic nodules and can be used diagnostically in fine-needle aspiration biopsies.

Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer

Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.

Comprehensive MicroRNA expression profiling identifies novel markers in follicular variant of papillary thyroid carcinoma

BACKGROUND Follicular variant of papillary thyroid carcinoma (FVPTC) shares features of papillary (PTC) and follicular (FTC) thyroid carcinomas on a clinical, morphological, and genetic level. MicroRNA (miRNA) deregulation was extensively studied in PTCs and FTCs. However, very limited information is available for FVPTC. The aim of this study was to assess miRNA expression in FVPTC with the most comprehensive miRNA array panel and to correlate it with the clinicopathological data. METHODS Forty-four papillary thyroid carcinomas (17 FVPTC, 27 classic PTC) and eight normal thyroid tissue samples were analyzed for expression of 748 miRNAs using Human Microarray Assays on the ABI 7900 platform (Life Technologies, Carlsbad, CA). In addition, an independent set of 61 tumor and normal samples was studied for expression of novel miRNA markers detected in this study. RESULTS Overall, the miRNA expression profile demonstrated similar trends between FVPTC and classic PTC. Fourteen miRNAs were deregulated in FVPTC with a fold change of more than five (up/down), including miRNAs known to be upregulated in PTC (miR-146b-3p, -146-5p, -221, -222 and miR-222-5p) and novel miRNAs (miR-375, -551b, 181-2-3p, 99b-3p). However, the levels of miRNA expression were different between these tumor types and some miRNAs were uniquely dysregulated in FVPTC allowing separation of these tumors on the unsupervised hierarchical clustering analysis. Upregulation of novel miR-375 was confirmed in a large independent set of follicular cell derived neoplasms and benign nodules and demonstrated specific upregulation for PTC. Two miRNAs (miR-181a-2-3p, miR-99b-3p) were associated with an adverse outcome in FVPTC patients by a Kaplan-Meier (p < 0.05) and multivariate Cox regression analysis (p < 0.05). CONCLUSIONS Despite high similarity in miRNA expression between FVPTC and classic PTC, several miRNAs were uniquely expressed in each tumor type, supporting their histopathologic differences. Highly upregulated miRNA identified in this study (miR-375) can serve as a novel marker of papillary thyroid carcinoma, and miR-181a-2-3p and miR-99b-3p can predict relapse-free survival in patients with FVPTC thus potentially providing important diagnostic and predictive value.

The involvement of miRNA Dysregulation in Amyotrophic Lateral Sclerosis

ALS is a neurodegenerative disease that specifically affects upper and lower motor neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutic approaches. Recent studies have shown that non-coding RNAs have a significant impact on normal CNS development and onset and progression of neurological disorders. Based on this evidence we specifically test the hypothesis that misregulation of miRNA expression is a common feature in familiar ALS. Hence, we are exploiting human neuroblastoma cell lines either expressing the SOD1(G93A) mutation or depleted from Fused in Sarcoma (FUS) as tools to investigate the role of miRNAs in familiar ALS. To this end we performed a genome-wide scale miRNA expression on these cells, using whole-genome small RNA deep-sequencing followed by quantitative real time validation (qPCR). This strategy allowed us to find a group of dysregulated miRNAs, which are predicted to play a role in the motorneurons physiology and pathology. We verified our data on cDNA derived from SOD1-ALS mice models at early stage of the disease and on cDNA derived from lymphocytes from a small group of ALS patients. In the future, we plan to define the mechanisms responsible for the miRNA dysregulation, by silencing or stimulating the signal transduction pathways putatively involved in miRNA expression and regulation.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down-regulated in primary AML patient samples and healthy CD34(+) progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.

An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

Strategies to determine the biological function of microRNAs

MicroRNAs (miRNAs) are regulators of gene expression that control many biological processes in development, differentiation, growth and metabolism. Their expression levels, small size, abundance of repetitive copies in the genome and mode of action pose unique challenges in studies elucidating the function of miRNAs. New technologies for identification, expression profiling and target gene validation, as well as manipulation of miRNA expression in vivo, will facilitate the study of their contribution to biological processes and disease. Such information will be crucial to exploit the emerging knowledge of miRNAs for the development of new human therapeutic applications.

Unravelling the impact of microRNA on Amyotrophic Lateral Sclerosis (ALS) pathogenesis

ALS is the most common adult neurodegenerative disease that specifically affects upper and lower neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutics. Recent studies have shown that noncoding RNA molecules have a significant impact on normal CNS development and on causes and progression of human neurological disorders. To investigate the hypothesis that expression of the mutant SOD1 protein, which is one of the genetic causes of ALS, may alter expression of miRNAs thereby contributing to the pathogenesis of familial ALS, we compared miRNA expression in SH-SY5Y expressing either the wild type or the SOD1 protein using small RNA deep-sequencing followed by RT-PCR validation. This strategy allowed us to find a group of up and down regulated miRNAs, which are predicted to play a role in the motorneurons physiology and pathology. The aim of my work is to understand if these modulators of gene expression may play a causative role in disease onset or progression. To this end I have checked the expression level of these misregulated miRNAs derived from RNA-deep sequencing by qPCR on cDNA derived from ALS mice models at early onset of the disease. Thus, I’m looking for the most up-regulated one even in Periferal Blood Mononuclear Cell (PBMC) of sporadic ALS patients. Furthermore I’m functionally characterizing the most up-regulated miRNAs through the validation of bioinformatic-predicted targets by analyzing endogenous targets levels after microRNA transfection and by UTR-report luciferase assays. Thereafter I’ll analyze the effect of misregulated targets on pathogenesis or progression of ALS by loss of functions or gain of functions experiments, based on the identified up/down-regulation of the specific target by miRNAs. In the end I would define the mechanisms responsible for the miRNAs level misregulation, by silencing or stimulating the signal transduction pathways putatively involved in miRNA regulation.

Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo.

BACKGROUND Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo. METHODS Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors. RESULTS miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo. CONCLUSIONS The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

MicroRNA profile of poorly differentiated thyroid carcinomas: new diagnostic and prognostic insights.

The diagnosis of conventional and oncocytic poorly differentiated (oPD) thyroid carcinomas is difficult. The aim of this study is to characterise their largely unknown miRNA expression profile and to compare it with well-differentiated thyroid tumours, as well as to identify miRNAs which could potentially serve as diagnostic and prognostic markers. A total of 14 poorly differentiated (PD), 13 oPD, 72 well-differentiated thyroid carcinomas and eight normal thyroid specimens were studied for the expression of 768 miRNAs using PCR-Microarrays. MiRNA expression was different between PD and oPD thyroid carcinomas, demonstrating individual clusters on the clustering analysis. Both tumour types showed upregulation of miR-125a-5p, -15a-3p, -182, -183-3p, -222, -222-5p, and downregulation of miR-130b, -139-5p, -150, -193a-5p, -219-5p, -23b, -451, -455-3p and of miR-886-3p as compared with normal thyroid tissue. In addition, the oPD thyroid carcinomas demonstrated upregulation of miR-221 and miR-885-5p. The difference in expression was also observed between miRNA expression in PD and well-differentiated tumours. The CHAID algorithm allowed the separation of PD from well-differentiated thyroid carcinomas with 73-79% accuracy using miR-23b and miR-150 as a separator. Kaplan-Meier and multivariate analysis showed a significant association with tumour relapses (for miR-23b) and with tumour-specific death (for miR-150) in PD and oPD thyroid carcinomas. MiRNA expression is different in conventional and oPD thyroid carcinomas in comparison with well-differentiated thyroid cancers and can be used for discrimination between these tumour types. The newly identified deregulated miRNAs (miR-150, miR-23b) bear the potential to be used in a clinical setting, delivering prognostic and diagnostic informations.

Circulating microRNA profiling in patients with advanced non-squamous NSCLC receiving bevacizumab/erlotinib followed by platinum-based chemotherapy at progression (SAKK 19/05).

OBJECTIVES Molecular subclassification of non small-cell lung cancer (NSCLC) is essential to improve clinical outcome. This study assessed the prognostic and predictive value of circulating micro-RNA (miRNA) in patients with non-squamous NSCLC enrolled in the phase II SAKK (Swiss Group for Clinical Cancer Research) trial 19/05, receiving uniform treatment with first-line bevacizumab and erlotinib followed by platinum-based chemotherapy at progression. MATERIALS AND METHODS Fifty patients with baseline and 24 h blood samples were included from SAKK 19/05. The primary study endpoint was to identify prognostic (overall survival, OS) miRNA's. Patient samples were analyzed with Agilent human miRNA 8x60K microarrays, each glass slide formatted with eight high-definition 60K arrays. Each array contained 40 probes targeting each of the 1347 miRNA. Data preprocessing included quantile normalization using robust multi-array average (RMA) algorithm. Prognostic and predictive miRNA expression profiles were identified by Spearman's rank correlation test (percentage tumor shrinkage) or log-rank testing (for time-to-event endpoints). RESULTS Data preprocessing kept 49 patients and 424 miRNA for further analysis. Ten miRNA's were significantly associated with OS, with hsa-miR-29a being the strongest prognostic marker (HR=6.44, 95%-CI 2.39-17.33). Patients with high has-miR-29a expression had a significantly lower survival at 10 months compared to patients with a low expression (54% versus 83%). Six out of the 10 miRNA's (hsa-miRN-29a, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a, hsa-miR-500a, hsa-miR-424) were insensitive to perturbations according to jackknife cross-validation on their HR for OS. The respective principal component analysis (PCA) defined a meta-miRNA signature including the same 6 miRNA's, resulting in a HR of 0.66 (95%-CI 0.53-0.82). CONCLUSION Cell-free circulating miRNA-profiling successfully identified a highly prognostic 6-gene signature in patients with advanced non-squamous NSCLC. Circulating miRNA profiling should further be validated in external cohorts for the selection and monitoring of systemic treatment in patients with advanced NSCLC.

Activation of common signaling pathways during remodeling of the heart and the bladder

The heart and the urinary bladder are hollow muscular organs, which can be afflicted by pressure overload injury due to pathological conditions such as hypertension and bladder outlet obstruction. This increased outflow resistance induces hypertrophy, marked by dramatic changes in the organs' phenotype and function. The end result in both the heart and the bladder can be acute organ failure due to advanced fibrosis and the subsequent loss of contractility. There is emerging evidence that microRNAs (miRNAs) play an important role in the pathogenesis of heart failure and bladder dysfunction. MiRNAs are endogenous non-coding single-stranded RNAs, which regulate gene expression and control adaptive and maladaptive organ remodeling processes. This Review summarizes the current knowledge of molecular alterations in the heart and the bladder and highlights common signaling pathways and regulatory events. The miRNA expression analysis and experimental target validation done in the heart provide a valuable source of information for investigators working on the bladder and other organs undergoing the process of fibrotic remodeling. Aberrantly expressed miRNA are amendable to pharmacological manipulation, offering an opportunity for development of new therapies for cardiac and bladder hypertrophy and failure.

miRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis

BACKGROUND Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. METHODS Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. RESULTS miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. CONCLUSIONS We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.