In vitro cross-talk between metastasis-competent circulating tumor cells and platelets in colon cancer: a malicious association during the harsh journey in the blood.

Background: Platelets are active players in hemostasis, coagulation and also tumorigenesis. The cross-talk between platelets and circulating tumor cells (CTCs) may have various pro-cancer effects, including promoting tumor growth, epithelial-mesenchymal transition (EMT), metastatic cell survival, adhesion, arrest and also pre-metastatic niche and metastasis formation. Interaction with CTCs might alter the platelet transcriptome. However, as CTCs are rare events, the cross-talk between CTCs and platelets is poorly understood. Here, we used our established colon CTC lines to investigate the colon CTC-platelet cross-talk in vitro and its impact on the behavior/phenotype of both cell types. Methods: We exposed platelets isolated from healthy donors to thrombin (positive control) or to conditioned medium from three CTC lines from one patient with colon cancer and then we monitored the morphological and protein expression changes by microscopy and flow cytometry. We then analyzed the transcriptome by RNA-sequencing of platelets indirectly (presence of a Transwell insert) co-cultured with the three CTC lines. We also quantified by reverse transcription-quantitative PCR the expression of genes related to EMT and cancer development in CTCs after direct co-culture (no Transwell insert) with platelets. Results: We observed morphological and transcriptomic changes in platelets upon exposure to CTC conditioned medium and indirect co-culture (secretome). Moreover, the expression levels of genes involved in EMT (p < 0.05) were decreased in CTCs co-cultured with platelets, but not of genes encoding mesenchymal markers (FN1 and SNAI2). The expression levels of genes involved in cancer invasiveness (MYC, VEGFB, IL33, PTGS2, and PTGER2) were increased. Conclusion: For the first time, we studied the CTC-platelet cross-talk using our unique colon CTC lines. Incubation with CTC conditioned medium led to platelet aggregation and activation, supporting the hypothesis that their interaction may contribute to preserve CTC integrity during their journey in the bloodstream. Moreover, co-culture with platelets influenced the expression of several genes involved in invasiveness and EMT maintenance in CTCs.

The Dynamics of miR-449a/c Expression during Uterine Cycles Are Associated with Endometrial Development.

The human endometrium is a highly dynamic tissue. Increasing evidence has shown that microRNAs (miRs) play essential roles in human endometrium development. Our previous assay, based on small RNA-sequencing (sRNA-seq) indicated the complexity and dynamics of numerous sequence variants of miRs (isomiRs) that can act together to control genes of functional relevance to the receptive endometrium (RE). Here, we used a greater average depth of sRNA-seq to detect poorly expressed small RNAs. The sequencing data confirmed the up-regulation of miR-449c and uncovered other members of the miR-449 family up-regulated in RE-among them miR-449a, as well as several isoforms of both miR-449a and miR-449c, while the third family member, miR-449b, was not identified. Stem-looped RT-qPCR analysis of miR expression at four-time points of the endometrial cycle verified the increased expression of the miR-449a/c family members in RE, among which the 5' isoform of miR-449c-miR-449c.1 was the most strongly up-regulated. Moreover, we found in a case study that the expression of miR-449c.1 and its precursor correlated with the histological assessment of the endometrial phase and patient age. We believe this study will promote the clinical investigation and application of the miR-449 family in the diagnosis and prognosis of human reproductive diseases.

Coupling miR/isomiR and mRNA Expression Signatures Unveils New Molecular Layers of Endometrial Receptivity.

Embryo implantation depends on endometrial receptivity (ER). To achieve ER, the preparation of the uterine lining requires controlled priming by ovarian hormones and the expression of numerous genes in the endometrial tissue. microRNAs (miRs) have emerged as critical genetic regulators of ER in fertility and of the diseases that are associated with infertility. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes can produce canonical miRs and variants-isomiRs. Here, we describe miR/isomiR expression dynamics across the four time points of natural chorionic gonadotropin (hCG)-administered cycles. Sequencing of the small RNAs (sRNA-seq) revealed that the most significant expression changes during the transition from the pre-receptive to the receptive phase occurred in the isomiR families of miR-125a, miR-125b, miR-10a, miR-10b, miR-449c, miR-92a, miR-92b, and miR-99a. Pairing the analysis of the differentially expressed (DE) miRs/isomiRs and their predicted DE mRNA targets uncovered 280 negatively correlating pairs. In the receptive endometrium, the 5'3'-isomiRs of miR-449c, which were among the most highly up-regulated isomiRs, showed a negative correlation with their target, transcription factor (TF) MYCN, which was down-regulated. Joint analysis of the miR/isomiR and TF expression identified several regulatory interactions. Based on these data, a regulatory TF-miR/isomiR gene-target circuit including let7g-5p and miR-345; the isomiR families of miR-10a, miR-10b, miR-92a, and miR-449c; and MYCN and TWIST1 was proposed to play a key role in the establishment of ER. Our work uncovers the complexity and dynamics of the endometrial isomiRs that can act cooperatively with miRs to control the functionally important genes that are critical to ER. Further studies of miR/isomiR expression patterns that are paired with those of their target mRNAs may provide a more in-depth picture of the endometrial pathologies that are associated with implantation failure.

miRGalaxy: Galaxy-Based Framework for Interactive Analysis of microRNA and isomiR Sequencing Data.

Tools for microRNA (miR) sequencing data analyses are broadly used in biomedical research. However, the complexity of computational approaches still remains a challenge for biologists with scarce experience in data analytics and bioinformatics. Here, we present miRGalaxy, a Galaxy-based framework for comprehensive analysis of miRs and their sequence variants-miR isoforms (isomiRs). Though isomiRs are commonly reported in deep-sequencing experiments, their detailed structure complexity and specific differential expression (DE) remain not fully examined by the majority of the available analysis tools. miRGalaxy encompasses biologist-user-friendly tools and workflows dedicated to the analysis of the isomiR-ome and its complex behavior in various biological samples. miRGalaxy is developed as a modular, accessible, redistributable, shareable, and user-friendly framework for scientists working with small RNA (sRNA)-seq data. Due to its modular workflow, advanced users can customize the steps and tools for their needs. In addition, the framework provides an analysis report where the significant output results are summarized in charts and visualizations. miRGalaxy can be accessed via preconfigured Docker image flavor and a Toolshed installation if the user already has a running Galaxy instance. Over the last decade, studies on the expression of miRs and isomiRs in normal and deregulated tissues have led to the discovery of their potential as diagnostic biomarkers. The detection of miRs in biofluids further expanded the exploration of the miR repertoire as a source of liquid biopsy biomarkers. Here we show the miRGalaxy framework application for in-depth analysis of the sRNA-seq data from two different biofluids, milk and plasma, to identify, annotate, and discover specific differentially expressed miRs and isomiRs.

Omics Analysis of Educated Platelets in Cancer and Benign Disease of the Pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is traditionally associated with thrombocytosis/hypercoagulation and novel insights on platelet-PDAC "dangerous liaisons" are warranted. Here we performed an integrative omics study investigating the biological processes of mRNAs and expressed miRNAs, as well as proteins in PDAC blood platelets, using benign disease as a reference for inflammatory noise. Gene ontology mining revealed enrichment of RNA splicing, mRNA processing and translation initiation in miRNAs and proteins but depletion in RNA transcripts. Remarkably, correlation analyses revealed a negative regulation on SPARC transcription by isomiRs involved in cancer signaling, suggesting a specific "education" in PDAC platelets. Platelets of benign patients were enriched for non-templated additions of G nucleotides (#ntaG) miRNAs, while PDAC presented length variation on 3' (lv3p) as the most frequent modification on miRNAs. Additionally, we provided an actionable repertoire of PDAC and benign platelet-ome to be exploited for future studies. In conclusion, our data show that platelets change their biological repertoire in patients with PDAC, through dysregulation of miRNAs and splicing factors, supporting the presence of de novo protein machinery that can "educate" the platelet. These novel findings could be further exploited for innovative liquid biopsies platforms as well as possible therapeutic targets.

isomiRs-Hidden Soldiers in the miRNA Regulatory Army, and How to Find Them?

Numerous studies on microRNAs (miRNA) in cancer and other diseases have been accompanied by diverse computational approaches and experimental methods to predict and validate miRNA biological and clinical significance as easily accessible disease biomarkers. In recent years, the application of the next-generation deep sequencing for the analysis and discovery of novel RNA biomarkers has clearly shown an expanding repertoire of diverse sequence variants of mature miRNAs, or isomiRs, resulting from alternative post-transcriptional processing events, and affected by (patho)physiological changes, population origin, individual's gender, and age. Here, we provide an in-depth overview of currently available bioinformatics approaches for the detection and visualization of both mature miRNA and cognate isomiR sequences. An attempt has been made to present in a systematic way the advantages and downsides of in silico approaches in terms of their sensitivity and accuracy performance, as well as used methods, workflows, and processing steps, and end output dataset overlapping issues. The focus is given to the challenges and pitfalls of isomiR expression analysis. Specifically, we address the availability of tools enabling research without extensive bioinformatics background to explore this fascinating corner of the small RNAome universe that may facilitate the discovery of new and more reliable disease biomarkers.