Highly sensitive and specific microRNA expression profiling using BeadArray technology

We have developed a highly sensitive, specific and reproducible method for microRNA (miRNA) expression profiling, using the BeadArray™ technology. This method incorporates an enzyme-assisted specificity step, a solid-phase primer extension to distinguish between members of miRNA families. In addition, a universal PCR is used to amplify all targets prior to array hybridization. Currently, assay probes are designed to simultaneously analyse 735 well-annotated human miRNAs. Using this method, highly reproducible miRNA expression profiles were generated with 100–200 ng total RNA input. Furthermore, very similar expression profiles were obtained with total RNA and enriched small RNA species (R² ≥ 0.97). The method has a 3.5–4 log (10⁵–10⁹ molecules) dynamic range and is able to detect 1.2- to 1.3-fold-differences between samples. Expression profiles generated by this method are highly comparable to those obtained with RT–PCR (R² = 0.85–0.90) and direct sequencing (R = 0.87–0.89). This method, in conjunction with the 96-sample array matrix should prove useful for high-throughput expression profiling of miRNAs in large numbers of tissue samples.     

Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs

Pseudomonas aeruginosa is a highly adaptable bacterium that thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen. This wide adaptability correlates with its broad genetic diversity. In this study, we used a deep-sequencing approach to explore the complement of small RNAs (sRNAs) in P. aeruginosa as the number of such regulatory molecules previously identified in this organism is relatively low, considering its genome size, phenotypic diversity and adaptability. We have performed a comparative analysis of PAO1 and PA14 strains which share the same host range but differ in virulence, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting. Interestingly, a number of these novel sRNAs are strain-specific or showed strain-specific expression, strongly suggesting that they could be involved in determining specific phenotypic traits.     

Identification of a common lupus disease-associated microRNA expression pattern in three different murine models of lupus

Background

Recent reports have shown that microRNAs (miRNAs) regulate vital immunological processes and have emerged as key regulators of immune system development and function. Therefore, it is important to determine miRNA dysregulation and its pathogenic contribution in autoimmune diseases, an aspect not adequately addressed thus far.

Methodology/Principal Findings

In this study, we profiled miRNA expressions in splenic lymphocytes from three murine lupus models (MRL-lpr, B6-lpr and NZB/W_F1) with different genetic background by miRNA microarray assays and Real-time RT-PCR. Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. The association of these miRNAs with the disease was highlighted by our observation that this miRNA expression pattern was evident in NZB/W mice only at an age when lupus disease is manifested. Further, we have shown that the miRNA dysregulation in MRL-lpr mice was not simply due to the activation of splenocytes. By Real-time RT-PCR, we confirmed that these miRNAs were upregulated in both purified splenic B and T cells from MRL-lpr mice. miR-127 and miR-379, which were greatly upregulated in splenocytes from lpr mice, were moderately increased in diseased NZB/W mice. In addition, Real-time RT-PCR revealed that miR-146a, miR-101a, and miR-17-92 were also markedly upregulated in splenic T, but not B cells from MRL-lpr mice.

Conclusions/Significance

The identification of common lupus disease-associated miRNAs now forms the basis for the further investigation of the pathogenic contribution of these miRNAs in autoimmune lupus, which will advance our knowledge of the role of miRNAs in autoimmunity. Given that miRNAs are conserved, with regard to both evolution and function, our observation of a common lupus disease-associated miRNA expression pattern in murine lupus models is likely to have significant pathogenic, diagnostic, and/or therapeutic implications in human lupus.     

Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.     

Comparative genomic analysis reveals evolutionary characteristics and patterns of microRNA clusters in vertebrates

MicroRNAs (miRNAs) are a class of small non-coding RNAs that can play important regulatory roles in many important biological processes. Although clustering patterns of miRNA clusters have been uncovered in animals, the origin and evolution of miRNA clusters in vertebrates are still poorly understood. Here, we performed comparative genomic analyses to construct 51 sets of orthologous miRNA clusters (SOMCs) across seven test vertebrate species, a collection of miRNA clusters from two or more species that are likely to have evolved from a common ancestral miRNA cluster, and used these to systematically examine the evolutionary characteristics and patterns of miRNA clusters in vertebrates. We found that miRNA clusters are continuously generated, and most of them tend to be conserved and maintained in vertebrate genomes, although some adaptive gains and losses of miRNA cluster have occurred during evolution. Furthermore, miRNA clusters appeared relatively early in the evolutionary history might suffer from more complicated adaptive gain-and-loss than those young miRNA clusters. Detailed analysis showed that genomic duplication events of ancestral miRNAs or miRNA clusters are likely to be major driving force and apparently contribute to origin and evolution of miRNA clusters. Comparison of conserved with lineage-specific miRNA clusters revealed that the contribution of duplication events for the formation of miRNA cluster appears to be more important for conserved miRNA clusters than lineage-specific. Our study provides novel insights for further exploring the origins and evolution of miRNA clusters in vertebrates at a genome scale.     

Real-time expression profiling of microRNA precursors in human cancer cell lines

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.     

Comparative analysis of mRNA isoform expression in cardiac hypertrophy and development reveals multiple post-transcriptional regulatory modules

Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the "fetal gene program". Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3'UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload.     

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach

Background

While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia.

Methodology/Principal Findings

A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue.

Conclusions/Significance

This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.     

MicroRNA regulation of molecular networks mapped by global microRNA, mRNA, and protein expression in activated T lymphocytes

MicroRNAs (miRNAs) regulate specific immune mechanisms, but their genome-wide regulation of T lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated, and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T lymphocytes) using locked nucleic acid inhibitors. Inhibition of these two highly upregulated miRNAs in CD4(+) T cells was shown to increase proliferation by removing suppression of four target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T lymphocyte immunity, underlining the value of mapping global gene, protein, and miRNA expression.