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Well-differentiated and dedifferentiated liposarcomas (WDLS/DDLS) account for approximately 13% of all soft tissue sarcoma in adults and cause substantial morbidity or mortality in the majority of patients. In this study, we evaluated the functions of miRNA (miR-193b) in liposarcoma in vitro and in vivo Deep RNA sequencing on 93 WDLS, 145 DDLS, and 12 normal fat samples demonstrated that miR-193b was significantly underexpressed in DDLS compared with normal fat. Reintroduction of miR-193b induced apoptosis in liposarcoma cells and promoted adipogenesis in human adipose-derived stem cells (ASC). Integrative transcriptomic and proteomic analysis of miR-193b-target networks identified novel direct targets, including CRK-like proto-oncogene (CRKL) and focal adhesion kinase (FAK). miR-193b was found to regulate FAK-SRC-CRKL signaling through CRKL and FAK. miR-193b also stimulated reactive oxygen species signaling by targeting the antioxidant methionine sulfoxide reductase A to modulate liposarcoma cell survival and ASC differentiation state. Expression of miR-193b in liposarcoma cells was downregulated by promoter methylation, resulting at least in part from increased expression of the DNA methyltransferase DNMT1 in WDLS/DDLS. In vivo , miR-193b mimetics and FAK inhibitor (PF-562271) each inhibited liposarcoma xenograft growth. In summary, miR-193b not only functions as a tumor suppressor in liposarcoma but also promotes adipogenesis in ASC. Furthermore, this study reveals key tyrosine kinase and DNA methylation pathways in liposarcoma, some with immediate implications for therapeutic exploration. Cancer Res; 77(21); 5728-40. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Central nervous system (CNS) is an increasingly common site of isolated metastasis for patients with Stage 4 neuroblastoma. To explore the microRNA (miRNA) profile of this metastatic process, miRNA sequencing was performed to identify miRNA sequence families with differential expression between tumor pairs (pre-CNS primary and CNS metastasis) from 13 patients with Stage 4 neuroblastoma. Seven miRNA sequence families had distinct expression in CNS metastases when compared with their corresponding pre-CNS primaries. MiR-7 was upregulated (3.75-fold), and miR-21, miR-22, miR-29a, miR-143, miR-199a-1-3p, and miR-199a-1-5p were downregulated (3.5-6.1-fold), all confirmed by quantitative reverse transcription-PCR. MiR-29a, previously shown to be downregulated in a broad spectrum of solid tumors including neuroblastoma, had the most significant decrease in all 13 CNS metastases (P = 0.001). Its known onco-targets CDC6, CDK6, and DNMT3A, as well as B7-H3, an inhibitory ligand for T cells, and natural killer cells, were found to have higher differential expression in these 13 CNS metastases when compared with their paired primaries. Additionally, miR-29a expression in primary tumors was significantly lower among patients who eventually relapsed in the CNS. Irrespective of the amplification status of MYCN, which is known to be associated with metastasis, pre-CNS primaries, and CNS metastases had significantly lower miR-29a expression than non-CNS primary tumors. Among MYCN amplified cell lines, those from CNS relapse also had lower miR-29a expression than non-CNS relapse. These findings raised the hypothesis that miR-29a could be a biomarker for neuroblastoma CNS metastasis, and its downregulation may play a pivotal role in CNS progression., (© 2014 Wiley Periodicals, Inc.)

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.

Background: Various 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., Results: To 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., Conclusions: We combined PAR-CLIP data with patient expression data to predict regulatory miRNAs, revealing potential therapeutic targets and prognostic markers in breast cancer.

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.

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.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

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.

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., (©2011 AACR.)

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., (©2011 AACR.)

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., (Copyright © 2011 AlphaMed Press.)

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.