Miguel Brown

MicroRNA Sequence and Expression Analysis in Breast Tumors by Deep Sequencing

MicroRNAs (miRNA) regulate many genes critical for tumorigenesis. We profiled miRNAs from 11 normal breast tissues, 17 noninvasive, 151 invasive breast carcinomas, and 6 cell lines by in-house-developed barcoded Solexa sequencing. miRNAs were organized in genomic clusters representing promoter-controlled miRNA expression and sequence families representing seed sequence-dependent miRNA target regulation. Unsupervised clustering of samples by miRNA sequence families best reflected the clustering based on mRNA expression available for this sample set. Clustering and comparative analysis of miRNA read frequencies showed that normal breast samples were separated from most noninvasive ductal carcinoma in situ and invasive carcinomas by increased miR-21 (the most abundant miRNA in carcinomas) and multiple decreased miRNA families (including miR-98/let-7), with most miRNA changes apparent already in the noninvasive carcinomas. In addition, patients that went on to develop metastasis showed increased expression of mir-423, and triple-negative breast carcinomas were most distinct from other tumor subtypes due to upregulation of the mir~17-92 cluster. However, absolute miRNA levels between normal breast and carcinomas did not reveal any significant differences. We also discovered two polymorphic nucleotide variations among the more abundant miRNAs miR-181a (T19G) and miR-185 (T16G), but we did not identify nucleotide variations expected for classical tumor suppressor function associated with miRNAs. The differentiation of tumor subtypes and prediction of metastasis based on miRNA levels is statistically possible but is not driven by deregulation of abundant miRNAs, implicating far fewer miRNAs in tumorigenic processes than previously suggested.

Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations

We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers.

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers

Significance Heart failure (HF) has a high morbidity and mortality and its incidence is increasing worldwide. While protein biomarkers have been established for diagnostic and prognostic evaluation of patients with HF, there is currently no systematic assessment of RNA biomarkers. We determined the composition of myocardial tissue and circulating microRNAs (miRNAs) in a large cohort of patients with stable and advanced HF and compared it to the composition of normal adult and fetal samples. The advanced HF patients underwent mechanical unloading with left ventricular assist devices and samples were collected at different postoperative time points. Our findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers. Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA–target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.

Human CLP1 Mutations Alter tRNA Biogenesis, Affecting Both Peripheral and Central Nervous System Function

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.

Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets.

BackgroundVarious microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to outcome in breast cancer, we integrated patient-paired miRNA-mRNA expression data with a set of validated miRNA targets and pathway inference.ResultsTo generate a biochemically-validated set of miRNA-binding sites, we performed argonaute-2 photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (AGO2-PAR-CLIP) in MCF7 cells. We then defined putative miRNA-target interactions using a computational model, which ranked and selected additional TargetScan-predicted interactions based on features of our AGO2-PAR-CLIP binding-site data. We subselected modeled interactions according to the abundance of their constituent miRNA and mRNA transcripts in tumors, and we took advantage of the variability of miRNA expression within molecular subtypes to detect miRNA repression. Interestingly, our data suggest that miRNA families control subtype-specific pathways; for example, miR-17, miR-19a, miR-25, and miR-200b show high miRNA regulatory activity in the triple-negative, basal-like subtype, whereas miR-22 and miR-24 do so in the HER2 subtype. An independent dataset validated our findings for miR-17 and miR-25, and showed a correlation between the expression levels of miR-182 targets and overall patient survival. Pathway analysis associated miR-17, miR-19a, and miR-200b with leukocyte transendothelial migration.ConclusionsWe combined PAR-CLIP data with patient expression data to predict regulatory miRNAs, revealing potential therapeutic targets and prognostic markers in breast cancer.

Multicolor microRNA FISH effectively differentiates tumor types

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues.

Bioinformatic analysis of barcoded cDNA libraries for small RNA profiling by next-generation sequencing

The characterization of post-transcriptional gene regulation by small regulatory RNAs of 20-30 nt length, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for the characterization of small RNAs is their identification and quantification across different developmental stages, normal and diseased tissues, as well as model cell lines. Here we present a step-by-step protocol for the bioinformatic analysis of barcoded cDNA libraries for small RNA profiling generated by Illumina sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.

Mammalian miRNA curation through next-generation sequencing.

Characteristic small RNA biogenesis processing patterns are used for the discovery of novel microRNAs (miRNAs) from next-generation sequencing data. Here, we highlight and discuss key criteria for mammalian – specifically human – miRNA database curation based on small RNA sequencing data. Sequence reads obtained from small RNA cDNA libraries are aligned to reference genomic regions, and miRNA genes are revealed by their distinct read length and bimodal read frequency distribution, the predicted secondary structure of the deduced miRNA stem-loop precursor molecule, and, to a lesser degree, based on evolutionary conservation of small RNAs from other vertebrates. Properly curated miRNA databases are an important resource for investigators interested in miRNA biology, diagnostics, and therapeutics.

Genome-wide annotation and analysis of zebra finch microRNA repertoire reveal sex-biased expression

BackgroundMicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally in a wide range of biological processes. The zebra finch (Taeniopygia guttata), an oscine songbird with characteristic learned vocal behavior, provides biologists a unique model system for studying vocal behavior, sexually dimorphic brain development and functions, and comparative genomics.ResultsWe deep sequenced small RNA libraries made from the brain, heart, liver, and muscle tissues of adult male and female zebra finches. By mapping the sequence reads to the zebra finch genome and to known miRNAs in miRBase, we annotated a total of 193 miRNAs. Among them, 29 (15%) are avian specific, including three novel zebra finch specific miRNAs. Many of the miRNAs exhibit sequence heterogeneity including length variations, untemplated terminal nucleotide additions, and internal substitution events occurring at the uridine nucleotide within a GGU motif. We also identified seven Z chromosome-encoded miRNAs. Among them, miR-2954, an avian specific miRNA, is expressed at significantly higher levels in males than in females in all tissues examined. Target prediction analysis reveals that miR-2954, but not other Z-linked miRNAs, preferentially targets Z chromosome-encoded genes, including several genes known to be expressed in a sexually dimorphic manner in the zebra finch brain.ConclusionsOur genome-wide systematic analysis of mature sequences, genomic locations, evolutionary sequence conservation, and tissue expression profiles of the zebra finch miRNA repertoire provides a valuable resource to the research community. Our analysis also reveals a miRNA-mediated mechanism that potentially regulates sex-biased gene expression in avian species.