Meike J. Saul

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E₂ (PGE₂) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E₂ synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE₂ biosynthesis. PGE₂ in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE₂ dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE₂ biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action.

Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.

Intercellular communication plays an essential role in lung cancer (LC). One of the major players in cell‐cell‐communication is small extracellular vesicles (sEV). SEV trigger various biological responses by transporting cellular cargo to target cells. One essential sEV component are microRNAs (miRs), whose transport has recently attracted increasing research interest. We report that prostaglandin E₂ (PGE₂), a key inflammatory lipid mediator, specifically induces the sorting of miR‐574‐5p in sEV of A549 and 2106T cells. We found that sEV‐derived miR‐574‐5p activates Toll‐like receptors (TLR) 7/8, thereby decreasing PGE₂‐levels. In contrast, intracellular miR‐574‐5p induces PGE₂‐biosynthesis. Consequently, the combination of intracellular and sEV‐derived miR‐574‐5p controls PGE₂‐levels via a feedback loop. This was only observed in adeno‐ but not in squamous cell carcinoma, indicating a cell‐specific response to sEV‐derived miRs, which might be due to unique tetraspanin compositions. Hence, we describe a novel function of miR‐574‐5p unique to adenocarcinoma. Intracellular miR‐574‐5p induces PGE₂ and thus the secretion of sEV‐derived miR‐574‐5p, which in turn decreases PGE₂‐biosynthesis in recipient cells.

Circulating microRNAs (miRs) represent promising diagnostic and prognostic biomarkers for various diseases. Despite the high number of biomarker studies, the results of these studies are hardly reproducible. This makes it difficult to transfer the results into clinical application. In this context, acceptance criteria for the quantification of miRs by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) may help to standardize miR analysis and improve reproducibility.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. Cell-derived small extracellular vesicles (sEV) mediate cell-to-cell communication in the synovial microenvironment by carrying microRNAs (miRs), a class of small non-coding RNAs. Herein, we report that sEV from synovial fluid promote osteoclast differentiation which is attributed to high levels of extracellular miR-574-5p. Moreover, we demonstrate for the first time that enhanced osteoclast maturation is mediated by Toll-like receptor (TLR) 7/8 signaling which is activated by miR-574-5p binding. This is a novel mechanism by which sEV and miRs contribute to RA pathogenesis and indicate that pharmacological inhibition of extracellular miR-574-5p might offer new therapeutic strategies to protect osteoclast-mediated bone destruction in RA.

MicroRNAs (miRs) are one of the most important post-transcriptional repressors of gene expression. However, miR-574-5p has recently been shown to positively regulate the expression of microsomal prostaglandin E-synthase-1 (mPGES-1), a key enzyme in the prostaglandin E2 (PGE2) biosynthesis, by acting as decoy to the RNA-binding protein CUG-RNA binding protein 1 (CUGBP1) in human lung cancer. MiR-574-5p exhibits oncogenic properties and promotes lung tumor growth in vivo via induction of mPGES-1 derived PGE2 synthesis. In a mass spectrometry-based proteomics study, we now attempted to characterize this decoy mechanism in A549 lung cancer cells at cellular level. Besides the identification of novel CUGBP1 targets, we identified that the interaction between miR-574-5p and CUGBP1specifically regulates mPGES-1 expression. This is supported by the fact that CUGBP1 and miR-574-5p are located in the nucleus, where CUGBP1 regulates alternative splicing. Further, in a bioinformatical approach we showed that the decoy-dependent mPGES-1 splicing pattern is unique. The specificity of miR-574-5p/CUGBP1 regulation on mPGES-1 expression supports the therapeutic strategy of pharmacological inhibition of PGE2 formation, which may provide significant therapeutic value for NSCLC patients with high miR-574-5p level.

Over the last years, many microRNAs (miRNAs) have been identified that regulate the formation of bioactive lipid mediators such as prostanoids and leukotrienes. Many of these miRNAs are involved in complex regulatory circuits necessary for the fine-tuning of biological functions including inflammatory processes or cell growth. A better understanding of these networks will contribute to the development of novel therapeutic strategies for the treatment of inflammatory diseases and cancer. In this review, we provide an overview of the current knowledge of miRNA regulation in eicosanoid pathways with special focus on novel miRNA functions and regulatory circuits of leukotriene and prostaglandin biosynthesis.

Extracellular microRNAs (miRs) have been proposed as important blood‐based biomarkers for several diseases. Contrary to proteins and other RNA classes, miRs are stable and easily detectable in body fluids. In this respect, miRs represent a perfect candidate for minimal invasive biomarkers which can hopefully become a complement for invasive histological examinations of tumor tissue. Despite the high number of miR biomarker studies, the specificity and reproducibility of these studies is missing. Therefore, the standardization of pre‐analytical and analytical methods is urgently needed. Here, we validated miR analysis for RNA isolation and miR quantification by quantitative polymerase chain reaction (RT‐qPCR) based on good laboratory practice (GLP). Validation was carried out exemplarily on four miRs, which had already been described as potential biomarkers in previous studies. As basis for RNA analysis using RT‐qPCR, the Minimum Information for Publication of Quantitative Real‐Time PCR Experiments were applied and adapted on the analysis of circulating miRs from human plasma. In our study, we identified and solved several pitfalls from handling to normalization strategy in the analysis of extracellular miRs that lead to inconsistent and non‐repeatable data. Principles of GLP set a framework of experimental design, performance and monitoring to ensure high quality and reliable data. Within this study, we appointed first acceptance criteria for circulating miR quantification during validation which set standards for future miR quantification in blood samples.

MicroRNAs (miRs) are small noncoding RNAs which control the expression of target genes by either translational repression or RNA degradation, known as canonical miR functions. The recent discovery that miR-328 has a noncanonical function and can activate gene expression by antagonizing the activity of heterogeneous ribonuclear protein E2 (hnRNP E2) opens an unexplored and exciting field of gene expression regulation. The global importance of such noncanonical miR function is not yet known. In order to achieve a better understanding of the new miR activity, we performed a compartment specific tandem mass tag (TMT)-based proteomic analysis in differentiated MonoMac6 (MM6) cells, to monitor gene expression variations in response to miR-328 knockdown. We identified a broad spectrum of novel potential miR-328/hnRNP E2 and miR-328 targets involved in regulation of compartment specific cellular processes, such as inflammation or RNA splicing. This study provides first insights of the global significance of noncanonical miR function.