Intra-individual variation of miRNA expression levels in human plasma samples

Background: Circulating miRNAs as potential non-invasive biomarkers for disease risk assessment and cancer early diagnosis have attracted increasing interest. Little information, however, is available regarding the intra-individual variation of circulating miRNA levels.

Methods: We measured expression levels of a panel of 800 miRNAs in repeated plasma samples from 51 healthy individuals that were collected 6 to 12?months apart and evaluated the intra-individual variation by the intra-class correlation coefficient (ICC).

Results: After background correction, a total of 185 miRNAs were detected in at least 10% of the plasma samples, with 69 and 28 miRNAs being detected in 50% and 90% of samples, respectively. The median ICC was 0.46 for these 185 miRNAs. Among them, 41% (75 miRNAs) had an ICC?≥?0.5, and 23% (42 miRNAs) had an ICC?≥?0.6. The ICC is higher for miRNAs with higher expression levels or higher detection rates, when compared to those with lower expression levels or lower detection rates.

Conclusions: These results suggest that common circulating miRNAs are stable over a relatively long period and can serve as reliable biomarkers for epidemiological and clinical research.     

Construction of miRNA-mRNA network for the identification of key biological markers and their associated pathways in IgA nephropathy by employing the integrated bioinformatics analysis

BackgroundAbout half-century ago, Immunoglobulin A nephropathy (IgAN) was discovered as a complicated disease with frequent clinical symptoms. Until now, exact mechanism underlying the pathogenesis of IgAN is poorly known. Therefore, current study was aimed to understand the molecular mechanism of IgAN by identifying the key miRNAs and their targeted hub genes. The key miRNAs might contribute to the diagnosis and therapy of IgAN, and could turn out to be a new star in the field of IgAN.MethodsThe microarray datasets were downloaded from Gene Expresssion Omnibus (GEO) database and analyzed using R package (LIMMA) in order to obtain differential expressed genes (DEGs). Then, the hub genes were identified using cytoHubba plugin of cytoscpae tool and other bioinformatics approaches including protein-protein interaction (PPI) network analysis, module analysis, and miRNA-hub gene network construction was also performed.ResultsA total of 348 DEGs were identified, of which 107 were upregulated genes and 241 were downregulated genes. Subsequently, the 12 overlapped genes were predicted from cytoHubba, and considered as hub genes. Moreover, a network among miRNA-hub genes was created to explore the correlation between the hub genes and their targeted miRNAs. Network construction ultimately lead to the identification of nine gene named FN1, EGR1, FOS, JUN, SERPINE1, MMP2, ATF3, MYC, and IL1B and one novel key miRNA namely, has-miR-144-3p as biomarker for diagnosis and therapy of IgAN.ConclusionThis study updates the information and yield a new perspective in context of understanding the pathogenesis and development of IgAN. In future, key miRNAs might be capable of improving the personalized detection and therapies for IgAN. In vivo and in vitro investigation of miRNAs and pathway interaction is essential to delineate the specific roles of the novel miRNAs, which may help to further reveal the mechanisms underlying IgAN.     

Role of morphine,miR-212/132 and mu opioid receptor in the regulation of Bdnf in zebrafish embryos

BackgroundMorphine is one of the first-line therapies for the treatment of pain despite its secondary effects. It modifies the expression of epigenetic factors like miRNAs. In the present study, we analyzed miR-212 and miR-132 and their implication in morphine effects in the zebrafish Central Nervous System (CNS) through the regulation of Bdnf expression.MethodsWe used control and knock-down zebrafish embryos to assess the effects of morphine in miRNAs 212/132 and mitotic or apoptotic cells by qPCR, immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB were studied by western blot and through a primary neuron culture. A luciferase assay was performed to confirm the binding of miRNAs 212/132 to mecp2.ResultsMorphine exposure decreases miR-212 but upregulates miR-132, as wells as Bdnf and TrkB, and changes the localization of proliferative cells. However, Bdnf expression was downregulated when miRNAs 212/132 and oprm1 were knocked-down. Furthermore, we proved that these miRNAs inhibit mecp2 expression by binding to its mRNA sequence. The described effects were corroborated in a primary neuron culture from zebrafish embryos.ConclusionsWe propose a mechanism in which morphine alters the levels of miRNAs 212/132 increasing Bdnf expression through mecp2 inhibition. oprm1 is also directly involved in this regulation. The present work confirms a relationship between the opioid system and neurotrophins and shows a key role of miR-212 and miR-132 on morphine effects through the regulation of Bdnf pathway.General significance.miRNAs 212/132 are novel regulators of morphine effects on CNS. Oprm1 controls the normal expression of Bdnf.     

Analysis of Glioblastoma Patients' Plasma Revealed the Presence of MicroRNAs with a Prognostic Impact on Survival and Those of Viral Origin

BackgroundGlioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.ResultsWe identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.ConclusionThe GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.     

In silico determination of transposon-derived miRNAs and targets in Aegilops species

Abstract

Transposable elements (TEs) are found almost in all living organism, shaping organisms’ genomes. miRNAs are noncoding RNA types which are especially important in gene expression regulations. Many previously determined plant miRNAs are identical/homologous to transposons (TE-MIR). The aim of this study was computational characterization of novel TE-related miRNAs and their targets in Aegilops genome by using stringent criteria. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed by BLAST2GO. Seventeen novel TE-related miRNAs in Aegilops genome were identified for the first time. GO analyses indicated that 40 targets played different roles in biological processes, cellular components and molecular functions. Moreover, these genes were involved in 10 metabolic pathways such as purine metabolism, nitrogen metabolism, oxidative phosphorylation, etc. as a result of KEGG analyses. Identification of miRNAs and their targets are significant to understand miRNA-TEs relationships and even how TEs affect plant growth and development. Obtaining results of this study are expected to provide possible new insight into Aegilops and its related species, wheat, with respect to miRNAs evolution and domestication.

Communicated by Ramaswamy H. Sarma     

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs

Background

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells.

Methods and results

polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells.

Conclusions

The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

     

Customized Internal Reference Controls for Improved Assessment of Circulating MicroRNAs in Disease

BackgroundAltered levels of circulating extracellular miRNA in plasma and serum have shown promise as non-invasive biomarkers of disease. However, unlike the assessment of cellular miRNA levels for which there are accepted housekeeping genes, analogous reference controls for normalization of circulating miRNA are lacking. Here, we provide an approach to identify and validate circulating miRNA reference controls on a de novo basis, and demonstrate the advantages of these customized internal controls in different disease settings. Importantly, these internal controls overcome key limitations of external spike-in controls.MethodsUsing a global RT-qPCR screen of 1066 miRNAs in plasma from pulmonary hypertension patients (PAH) and healthy subjects as a case example, we identified a large pool of initial candidate miRNAs that were systematically ranked according to their plasma level stability using a predefined algorithm. The performance of the top candidates was validated against multiple comparators, and in a second independent cohort of PAH and control subjects. The broader utility of this approach was demonstrated in a completely different disease setting with 372 miRNAs screened in plasma from septic shock patients and healthy controls.ResultsNormalization of data with specific internal reference controls significantly reduced the overall variation in circulating miRNA levels between subjects (relative to raw data), provided a more balanced distribution of up- and down-regulated miRNAs, replicated the results obtained by the benchmark geometric averaging of all detected miRNAs, and outperformed the commonly used external spike-in strategy.ConclusionsWe demonstrate the feasibility of identifying circulating reference controls that can reduce extraneous technical variations, and improve the assessment of disease-related changes in plasma miRNA levels. This study provides a novel conceptual framework that addresses a critical and previously unmet need if circulating miRNAs are to advance as reliable diagnostic tools in medicine.     

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0568-y) contains supplementary material, which is available to authorized users.     

Protein network-based Lasso regression model for the construction of disease-miRNA functional interactions

BackgroundThere is a growing body of evidence associating microRNAs (miRNAs) with human diseases. MiRNAs are new key players in the disease paradigm demonstrating roles in several human diseases. The functional association between miRNAs and diseases remains largely unclear and far from complete. With the advent of high-throughput functional genomics techniques that infer genes and biological pathways dysregulted in diseases, it is now possible to infer functional association between diseases and biological molecules by integrating disparate biological information.ResultsHere, we first used Lasso regression model to identify miRNAs associated with disease signature as a proof of concept. Then we proposed an integrated approach that uses disease-gene associations from microarray experiments and text mining, and miRNA-gene association from computational predictions and protein networks to build functional associations network between miRNAs and diseases. The findings of the proposed model were validated against gold standard datasets using ROC analysis and results were promising (AUC=0.81). Our protein network-based approach discovered 19 new functional associations between prostate cancer and miRNAs. The new 19 associations were validated using miRNA expression data and clinical profiles and showed to act as diagnostic and prognostic prostate biomarkers. The proposed integrated approach allowed us to reconstruct functional associations between miRNAs and human diseases and uncovered functional roles of newly discovered miRNAs.ConclusionsLasso regression was used to find associations between diseases and miRNAs using their gene signature. Defining miRNA gene signature by integrating the downstream effect of miRNAs demonstrated better performance than the miRNA signature alone. Integrating biological networks and multiple data to define miRNA and disease gene signature demonstrated high performance to uncover new functional associations between miRNAs and diseases.     

MicroRNAs as Potential Diagnostic New Biomarkers in Diagnosis of Sepsis in Pediatric Patients

Background:Sepsis is one of the most common causes of multiorgan failure. Sepsis requires the presence of infection with a resultant systemic inflammatory state. Organ dysfunction occurs from the combination of the two processes. Sepsis is the main cause of mortality at intensive care units, with 30-50% mortality rate for all septic episodes. MicroRNA (miRNA) profile data could be beneficial as a specific diagnostic biomarker for sepsis and systemic inflammatory response syndrome (SIRS).Methods:Expression of miRNAs -122, -181b, -223 and -146a levels were assayed by quantitative real time polymerase chain reaction (qRT-PCR) in a prospective case control study, where forty septic cases were compared to 40 healthy controls of matched age and gender. Results:miRNAs -122 and -181b were significantly upregulated during early septic conditions, indicating that they could be sensitive and specific biomarkers for diagnosing sepsis. miRNA-223 and miRNA-146a could also represent highly specific and sensitive diagnostic biomarkers, as they were found to be significantly down-regulated. Serum levels of miRNA-223 could be used to predict poor prognosis with 70% sensitivity and 75% specificity, whereas the other three miRNAs could not predict prognosis.Conclusion:Our study shows that all tested miRNAs can be used for early detection of sepsis, with miRNA-223 being predictive of mortality, hence preventing multi-organ failure and reducing mortality, and predicting poor outcomes, thereby assisting in early categorization of ICU patients for rapid appropriate treatment and medico legal aspects.Key Words: Micrna-223, Systemic inflammatory response syndrome (SIRS), Sepsis, Biomarker     

A panel of three plasma microRNAs for colorectal cancer diagnosis

BackgroundDifferential microRNA (miRNA) expression profiles in plasma or serum were identified, providing foundation for studying their potentially diagnostic role in colorectal cancer (CRC).MethodsWe performed S-poly(T) Plus PCR assay to select and validate differentially expressed plasma miRNAs from a sample set including 101 CRC patients, 20 patients with colorectal noncancerous polyps (NCP), and 134 healthy controls. And bioinformatics methods was used to integrated predicted or validated targets of the differentially dysregulated miRNAs and analyzed their overrepresented pathways.ResultsAfter the two-phase selection and validation process, we identified a miRNA panel (miR-144-3p, miR-425-5p, and miR-1260b) with high diagnostic efficiency for CRC; the panel distinguished CRC patients from controls with 93.8% sensitivity and 91.3% specificity. Results indicated that the dysregulated miRNAs in CRC were functionally involved in several key cancer-related pathways, such as axonal guidance, PI3K, and calcium signaling pathways.ConclusionsOur study demonstrated that a plasma 3-miRNA panel may serve as a novel noninvasive biomarker to diagnose CRC. This plasma 3-miRNA panel may be related to CRC development. However, further studies are needed to highlight its theoretical strengths.     

中华蜜蜂幼虫肠道响应球囊菌胁迫的microRNA应答分析

【目的】蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)是一种能够侵染中华蜜蜂(Apis cerana cerana,简称中蜂)幼虫的致死性真菌病原。微小RNA(microRNA,miRNA)可通过在转录后水平靶向抑制或降解mRNA而参与宿主与病原互作过程。本研究旨在对球囊菌胁迫的中蜂6日龄幼虫肠道的差异表达miRNA(DEmiRNA)及其靶基因进行深入分析,进而揭示DEmiRNA在中蜂响应球囊菌胁迫应答过程中的作用。【方法】利用Illumina MiSeq平台对正常及球囊菌胁迫的中蜂6日龄幼虫肠道(AcCK和AcT)进行测序,通过相关生物信息学软件预测DEmiRNA及其靶基因。通过Blast将靶基因注释到GO和KEGG数据库。利用Cytoscape软件构建DEmiRNA与其靶mRNA的调控网络。通过Stem-loop RT-PCR和qPCR验证测序数据的可靠性。【结果】本研究共预测出537个miRNA,其长度分布介于16–35 nt之间,且不同长度的miRNA首位碱基偏向性差异明显。通过Stem-loop RT-PCR证实了10个novel miRNA的表达。AcCK vs AcT比较组共有54个DEmiRNA,包含31个上调和23个下调miRNA,可分别靶向结合6170和8199个靶基因。GO分类结果显示上调和下调miRNA的靶基因分别涉及47和47个条目,富集基因数最多的皆为结合细胞进程和催化活性。KEGG代谢通路(pathway)富集分析结果表明上调和下调miRNA的靶基因分别富集在134和126条pathway,富集基因数最多的均为内吞作用和内质网中的蛋白质加工。调控网络分析结果表明,DEmiRNA及其靶mRNA形成十分复杂的调控关系;31个DEmiRNA可靶向结合51个与泛素介导的蛋白水解相关的mRNA,18个DEmiRNA可靶向结合14个与Jak-STAT信号通路相关的mRNA;miR-1277-x、miR-26-x、miR-27-y、miR-30-x、miR-6052-x等16个miRNA共同参与了上述两条免疫通路的调控。最后,随机挑选3个DEmiRNA进行qPCR验证,结果证明了测序数据的可靠性。【结论】本研究提供了中蜂幼虫肠道在球囊菌胁迫后期的miRNA的表达谱和差异表达信息,揭示了球囊菌与宿主之间在miRNA组学水平存在复杂的互作。miR-6052-x和miR-1277-x作为调控网络的核心可能通过影响细胞凋亡参与宿主的免疫防御,miR-26-x和miR-30-x可能通过调控Jak-STAT信号通路参与宿主的胁迫应答。本研究筛选出的关键DEmiRNA有望作为治疗白垩病的分子靶标。     

外泌体miRNA生物标志物在肝癌中的研究进展*

外泌体广泛存在于多种体液中,携带有大量活性物质,如mRNA、miRNA、蛋白和脂质等。其中的miRNA是一类短非编码RNA,在转录后水平调节基因的表达,广泛参与个体生长发育等各生命活动。外泌体miRNA有多种生物学功能,在肿瘤的发生发展、侵袭转移、机体耐药及免疫调控等多方面发挥着重要作用。目前的研究表明,无论是作为肿瘤早筛早诊和预后评估标志物还是用于肿瘤治疗,外泌体miRNA都有很好的应用前景。本文就近年来外泌体miRNA在肝癌中的研究进展和临床应用进行综述。     

Diverse MicroRNAs‐mRNA networks regulate the priming phase of mouse liver regeneration and of direct hyperplasia

ObjectivesAdult hepatocytes are quiescent cells that can be induced to proliferate in response to a reduction in liver mass (liver regeneration) or by agents endowed with mitogenic potency (primary hyperplasia). The latter condition is characterized by a more rapid entry of hepatocytes into the cell cycle, but the mechanisms responsible for the accelerated entry into the S phase are unknown.Materials and methodsNext generation sequencing and Illumina microarray were used to profile microRNA and mRNA expression in CD‐1 mice livers 1, 3 and 6 h after 2/3 partial hepatectomy (PH) or a single dose of TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Ingenuity pathway and DAVID analyses were performed to identify deregulated pathways. MultiMiR analysis was used to construct microRNA‐mRNA networks.ResultsFollowing PH or TCPOBOP we identified 810 and 527 genes, and 102 and 10 miRNAs, respectively, differentially expressed. Only 20 genes and 8 microRNAs were shared by the two conditions. Many miRNAs targeting negative regulators of cell cycle were downregulated early after PH, concomitantly with increased expression of their target genes. On the contrary, negative regulators were not modified after TCPOBOP, but Ccnd1 targeting miRNAs, such as miR‐106b‐5p, were downregulated.ConclusionsWhile miRNAs targeting negative regulators of the cell cycle are downregulated after PH, TCPOBOP caused downregulation of miRNAs targeting genes required for cell cycle entry. The enhanced Ccnd1 expression may explain the more rapid entry into the S phase of mouse hepatocytes following TCPOBOP.