Aleksandra Mihailovic

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.

miRNA in situ hybridization in formaldehyde and EDC–fixed tissues

MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5′ phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.

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.

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area.

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.

Small RNA sequencing and functional characterization reveals microRNA-143 tumor suppressor activity in liposarcoma

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21 and miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143 and miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, topoisomerase 2A, protein regulator of cytokinesis 1 (PRC1), and polo-like kinase 1 (PLK1). The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G(2)-M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma.

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells.

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor‐matched CD133+ cells with the more differentiated CD34+CD133− and CD34−CD133− cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing‐based profiling, we found that between 109 (CD133+) and 216 (CD34−CD133−) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR‐142‐3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34+CD133− cells. Eighteen miRNAs were significantly differentially expressed between CD133+ and CD34+CD133− cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase‐based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR‐142‐3p as well as tropomyosin 1 and frizzled homolog 5 by miR‐29a. Overexpression of miRNAs in CD133+ cells demonstrated that miR‐142‐3p has a negative influence on the overall colony‐forming ability. In conclusion, the miRNAs expressed differentially between the CD133+ and CD34+CD133− cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell‐based therapies with CD133+ cells and delineate for the first time how the stem cell character of CD133+ cells is defined by the expression of specific miRNAs. STEM CELLS 2011;29:847–857

Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

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

The miR-17-92 cluster and its target THBS1 are differentially expressed in angiosarcomas dependent on MYC amplification.

Angiosarcomas (ASs) represent a heterogeneous group of malignant vascular tumors that may occur spontaneously as primary tumors or secondarily after radiation therapy or in the context of chronic lymphedema. Most secondary ASs have been associated with MYC oncogene amplification, whereas the role of MYC abnormalities in primary AS is not well defined. Twenty‐two primary and secondary ASs were analyzed by array‐comparative genomic hybridization (aCGH) and by deep sequencing of small RNA libraries. By aCGH and subsequently confirmed by fluorescence in situ hybridization, MYC amplification was identified in three out of six primary tumors and in 8 out of 12 secondary AS. We have also found MAML1 as a new potential oncogene in MYC‐amplified AS. Significant upregulation of the miR‐17‐92 cluster was observed in MYC‐amplified AS compared to AS lacking MYC amplification and the control group (other vascular tumors, nonvascular sarcomas). Moreover, MYC‐amplified ASs were associated with a significantly lower expression of thrombospondin‐1 (THBS1) than AS without MYC amplification or controls. Altogether, our study implicates MYC amplification not only in the pathogenesis of secondary AS but also in a subset of primary AS. Thus, MYC amplification may play a crucial role in the angiogenic phenotype of AS through upregulation of the miR‐17‐92 cluster, which subsequently downregulates THBS1, a potent endogenous inhibitor of angiogenesis.

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.