Project description:Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these 4 were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions.

Project description:Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of progressive and genetic neuromuscular disorders that involve weakness and wasting of predominantly pelvic and shoulder girdle muscles. More than 30 subtypes have been identified, with variable phenotype. In the early stages of the disease, these LGMD share common clinical and histopathological characteristics among different subtypes and with other neuromuscular disorders, complicating the disease-subtype identification and thus, lengthening the final diagnosis of the disease. In the present study, we try to identify by a non-invasive method a molecular signature including biochemical and epigenetic parameters with a potential value for LGMD patient prognosis and stratification. Circulating miRNome was obtained by smallRNA-seq in plasma samples from LGMD patients (n=6) and matched controls (n=6). Over-representation analysis (ORA) using GO terms and KEGG database was used to study the functional enrichment of target genes. Biochemical parameters related to calcium metabolism, hormonal status, and muscle metabolism were also analyzed in these patients. Thirteen differentially expressed miRs (FDR < 0.05) were able to separate each clinical group by hierarchical clustering. Indeed, most of differentially expressed miRs were up-regulated. Functional enrichment analysis of the target genes for the dysregulated miRs revealed that targets of up-regulated miRs modulated cell cycle and cancer related pathways, while targets of down-regulated miRs were involved in muscle tissue development, regeneration and senescence. Four of the circulating miRs (miR-19b-3p, miR-192-5p, miR-122-5p and miR 323-3p) were further validated by qPCR in LGMD and in the more frequent muscular dystrophies: Duchenne (DMD)(n=5) and facioscapulohumeral muscular dystrophy (FSHD)(n=4). Receiver operating characteristic curve analysis revealed high AUC values for selected miRs (1.0 for miR-19b-3p, miR-192-5p and miR-122-5p; 0.75 for miR-323-3p) suggesting that these miRs could be good biomarker candidates for LGMD. The covariates gender, age at disease onset, and ambulation did not correlate with the expression levels of these miRs. However, differential expression of miR-122-5p (≥10-60 fold, p< 0.0001), miR-192-5p (≥10-60 fold, p< 0.0001) and miR-323-3p (≤ 2-10 fold, p<0.05) levels compared to matched controls could discriminate LGMD from DMD or FSHD dystrophies. In addition, a strong correlation between some of these circulating miRs and biochemical variables such as CK, vitamin D3, ALP and PTH was found only in LGMD patients.

Project description:Bi-directional communication with the microenvironment is essential for homing and survival of cancer cells with implications for disease biology and behaviour. In chronic lymphocytic leukemia (CLL) the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly characterised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originating from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50 - 100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosomes-specific and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p impacts on the expression of 'suppressor of fused' (Sufu), a Hedgehog (Hh) signalling intermediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology. miR analysis was carried out on 3 exosomal samples and 3 corresponding cellular samples from CLL patients. Exosomes are a discrete population of small (50-100 nm diameter) EVs of endosomal origin with a lipid membrane bilayer and a cup-shaped morphology. We used common reference design which allows visualization of variations between the samples. Each sample equals one array and each sample compared with common reference (Pool). The pool or common reference was a collection of 23 RNA samples isolated from 23 CLL cases. We hypothesised that CLL derived exosomes should contain unique miRs that are reflective of CLL cell content and additionally relevant for disease biology.

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Viral miRNAs alter cellular miRNAs profiles

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