Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index-the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10(-16)). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10(-7)). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10(-37)). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10(-22)), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10(-6)). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such as female gender, age at onset and severity. This is the largest study population so far investigated for the genetic influence on antibody levels in the cerebrospinal fluid in multiple sclerosis, including 6950 patients. We confirm that genetic factors underlie these antibody levels and identify both the major histocompatibility complex and immunoglobulin heavy chain region as major determinants.
Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids, and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes are still not completely elucidated. In this study, by the use of next-generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and noncoding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis for studying the involvement of RNA-binding proteins capable of recognizing RNA sequences and their role in the export of RNAs into exosomes.
Minimally-invasive tools to assess tumour presence and burden may improve clinical management. FDG-PET (metabolic) imaging is the current gold standard for interim response assessment in patients with classical Hodgkin Lymphoma (cHL), but this technique cannot be repeated frequently. Here we show that microRNAs (miRNA) associated with tumour-secreted extracellular vesicles (EVs) in the circulation of cHL patients may improve response assessment. Small RNA sequencing and qRT-PCR reveal that the relative abundance of cHL-expressed miRNAs, miR-127-3p, miR-155-5p, miR-21-5p, miR-24-3p and let-7a-5p is up to hundred-fold increased in plasma EVs of cHL patients pre-treatment when compared to complete metabolic responders (CMR). Notably, in partial responders (PR) or treatment-refractory cases (n = 10) the EV-miRNA levels remain elevated. In comparison, tumour specific copy number variations (CNV) were detected in cell-free DNA of 8 out of 10 newly diagnosed cHL patients but not in patients with PR. Combining EV-miR-127-3p and/or EV-let-7a-5p levels, with serum TARC (a validated protein cHL biomarker), increases the accuracy for predicting PET-status (n = 129) to an area under the curve of 0.93 (CI: 0.87-0.99), 93.5% sensitivity, 83.8/85.0% specificity and a negative predictive value of 96%. Thus the level of tumour-associated miRNAs in plasma EVs is predictive of metabolic tumour activity in cHL patients. Our findings suggest that plasmaThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
The chemotherapeutic drug epirubicin increases the exosomal export of miR-503 in endothelial cells. To understand the mechanisms behind this process, we transfected endothelial cells with miR-503 carrying a biotin tag. Then, we pulled-down the proteins interacting with miR-503 and studied their role in microRNA exosomal export. A total of four different binding partners were identified by mass spectrometry and validated by western blotting and negative controls, among them ANXA2 and hnRNPA2B1. Using knock-down systems combined with pull-down analysis, we determined that epirubicin mediates the export of miR-503 by disrupting the interaction between hnRNPA2B1 and miR-503. Then, both ANXA2 and miR-503 are sorted into exosomes while hnRNPA2B1 is relocated into the nucleus. The combination of these processes culminates in the increased export of miR-503. These results suggest, for the first time, that RNA-binding proteins can negatively regulate the exosomal sorting of microRNAs. Keywords Exosomes • EVs • MicroRNAs • Exosomal export • RNA-binding proteins Abbreviations Epi Epirubicin EVs Extracellular vesicles HUVECs Human umbilical vein endothelial cells HMVECs Human microvascular endothelial cells ANXA2 Annexin A2 hnRNPA2B1 Heteronuclear ribonucleoprotein A2/B1 TSP1/thbs1 Thrombospondin 1 SYN Syntenin VIM Vimentin Vinculin Vinculin MEC MicroRNA exporting complex Cellular and Molecular Life Sciences Jennifer Pérez-Boza and Amandine Boeckx contributed equally to the work.
Epithelial-mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3′-UTR-luciferase reporter vector assays implicated the 3′-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3′UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.
The analysis of biomarkers in biological fluids, also known as liquid biopsies, is seen with great potential to diagnose complex diseases such as cancer with a high sensitivity and minimal invasiveness. Although it can target any biomolecule, most liquid biopsy studies have focused on circulating nucleic acids. Historically, studies have aimed at the detection of specific mutations on cell-free DNA (cfDNA), but recently, the study of cell-free RNA (cfRNA) has gained traction. Since 2020, a handful of cfDNA tests have been approved for therapy selection by the FDA, however, no cfRNA tests are approved to date. One of the main drawbacks in the field of RNA-based liquid biopsies is the low reproducibility of the results, often caused by technical and biological variability, a lack of standardized protocols and insufficient cohorts. In this review, we will identify the main challenges and biases introduced during the different stages of biomarker discovery in liquid biopsies with cfRNA and propose solutions to minimize them.
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