ABSTRACT: Pancreatic β-cell dysfunction caused by obesity can be associated with alterations in the levels of microRNAs (miRNAs). However, the role of miRNAs in such processes remains elusive. Here, we show that pancreatic islet miR-27a-5p, which is markedly increased in obese mice and impairs insulin secretion, is mainly delivered by visceral adipocyte-derived extracellular vesicles (EVs). Depleting miR-27a-5p significantly improves insulin secretion and glucose intolerance in db/db mice. Supporting the function of EVs’ miR-27a-5p as a key pathogenic factor, intravenous injection of miR-27a-5p-containing EVs shows their distribution in mouse pancreatic islets. Tracing the injected AAV-miR-27a-5p (AAV-miR-27a) or AAV-FABP4-miR-27a-5p (AAV-FABP4-miR-27a) in visceral fat results in upregulating miR-27a-5p in EVs and serum, and elicits mouse pancreatic β-cell dysfunction. Mechanistically, miR-27a-5p directly targets L-type Ca2+ channel subtype CaV1.2 (Cacna1c) and reduces insulin secretion in β-cells. Overexpressing mouse CaV1.2 largely abolishes the insulin secretion injury induced by miR-27a-5p. These findings reveal a causative role of EVs’ miR-27a-5p in visceral adipocyte-mediated pancreatic β-cell dysfunction in obesity-associated type 2 diabetes mellitus.
Project description:Epidemiological evidence has identified an association between breast cancer (BC) and systemic dysregulation of glucose metabolism. However, how BC influences glucose homeostasis remains unknown. Here we show that BC-derived extracellular vesicles (EVs) suppress pancreatic endocrine secretion to systemically reset glucose homeostasis. In pancreatic β-cells, miR-122 delivered in BC-derived EVs targets PKM to suppress glycolysis and ATP-dependent insulin exocytosis. Mice receiving high-miR-122 EVs or bearing BC xenograft tumors, but not those with tumors deficient in EV secretion or miR-122, exhibit suppressed insulin secretion, enhanced endogenous glucose production, impaired glucose tolerance, and hyperglycemia. Compared to non-cancer controls, BC patients have higher levels of EV-encapsulated miR-122 and fasting glucose but lower insulin levels in blood; the miR-122 levels are positively associated with glucose and negatively associated with insulin. This EV-mediated glucose reallocation at the whole-body level may contribute to tumor growth and progression, as well as higher incidence of diabetes in BC patients.
Project description:MicroRNAs (miRNAs) are small non-coding RNAs that function as modulators of gene expression. We previously showed that miR-146a-5p is upregulated in pancreatic islets treated with pro-inflammatory cytokines and in pancreatic sections from organ donors with type 1 diabetes (T1D). Other studies have associated overexpression of miR-146a-5p with β cell apoptosis and impaired insulin secretion; however, the molecular mechanisms mediating these effects remain elusive. To investigate the role of miR-146a-5p in β cell function, we developed stable MIN6 cell lines transduced with lentiviral vectors to either overexpress or inhibit the expression of miR-146a-5p. Monoclonal cell populations were treated with pro-inflammatory cytokines (IL1β, IFNg, and TNFα) to model T1D in vitro. We found that overexpression of miR-146a-5p increased the cell death of MIN6 cells under inflammatory stress, whereas inhibition of miR-146a-5p reversed these effects. Additionally, inhibition of miR-146a-5p increased mitochondrial DNA copy number, respiration rate, and ATP production, suggesting that miR-146a-5p inhibition improves mitochondrial function. In support of this finding, we also observed that miR-146a-5p is enriched in the mitochondria of MIN6 cells treated with cytokines. Consistently, bioinformatic analysis of RNA sequencing data using MIN6 stable cells showed enrichment of pathways related to insulin secretion, apoptosis, and mitochondrial function when the expression levels of miR-146a-5p were altered. Overall, the findings from our study show for the first time that miR-146a-5p upregulation during inflammatory stress may promote β cell dysfunction and death by suppressing mitochondrial function.
Project description:We have recently confirmed miR-27a-3p as a crucial regulator of human adipogenesis (Wu H, Pula T, Tews D, Amri E-Z, Debatin K-M, Wabitsch M, Fischer-Posovszky P, Roos J. microRNA-27a-3p but Not -5p Is a Crucial Mediator of Human Adipogenesis. Cells. 2021; 10(11):3205. https://doi.org/10.3390/cells10113205 ). MiR-27a-5p did not impair human adipogenesis. However, since several publications state that miR-27a ist also a crucial regulator of UCP1, we were interested if miR-27a-3p or miR-27a-5p regulatas UCP1 and other thermogenesis related genes. We found a strong regulation of UCP1 with functional relevance for the cellular metabolism by miR-27a-5p.To asesse the mRNA gene expression pattern, mRNA sequencing was performed.
Project description:Human NK cells activity against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. In order to identify possible mechanisms mediating chemokine receptors modulation, we analyzed the miRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3’ untranslated region (3’UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3’UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p down-regulates the surface expression of CX3CR1. Finally we showed that Neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX3CR1 expression.
Project description:Pancreatic β cell dysfunction greatly contributes to the pathogenesis of type 2 diabetes. MiR-21 has been shown to be induced in the islets of glucose intolerant patients and type 2 diabetic mice. However, the role of miR-21 in the regulation of pancreatic β cell function remains largely elusive. In the current study, we studied the pathway by which miR-21 regulates glucose-stimulated insulin secretion utilizing mice lacking miR-21 in their β cells (miR-21βKO). We found that miR-21βKO mice developed glucose intolerance due to impaired glucose-stimulated insulin secretion. Mechanistic studies revealed that miR-21 enhances glucose uptake and subsequently promotes insulin secretion by up-regulating Glut2 expression in a miR-21-Pdcd4-AP-1 dependent pathway. Over-expression of Glut2 in knockout islets resulted in rescue of the impaired glucose-stimulated insulin secretion. Furthermore, we demonstrated that delivery of miR-21 into the pancreas of type 2 diabetic db/db mice is able to promote Glut2 expression and significantly reduce blood glucose level. Taking together, our results reveal that miR-21 in islet β cell promotes insulin secretion and support a role for miR-21 in the adaptation of pancreatic β cell function in type 2 diabetes.
Project description:MiRNAs have been shown to alter both protein expression and secretion in different cellular contexts. By combining in vitro, in vivo and in silico techniques, we demonstrated that overexpression of pre-miR-1307 reduced the ability of breast cancer cells to induce endothelial cell sprouting and angiogenesis. However, the molecular mechanism behind this and the effect of the individual mature miRNAs derived from pre-miR-1307 on protein secretion and is largely unknown. Here, we overexpressed miR-1307-3p|0, -3p|1 and 5p|0 in MDA-MB-231 breast cancer cells and assessed the impact of miRNA overexpression on protein secretion by Mass Spectrometry. Unsupervised hierarchical clustering revealed a distinct phenotype induced by overexpression of miR-1307-5p|0 compared to the controls and to the 5’isomiRs derived from the 3p-arm. Together, our results suggest different impacts of miR-1307-3p and miR-1307-5p on protein secretion which is in line with our in vitro observation that miR-1307-5p, but not the isomiRs derived from the 3p-arm reduce endothelial cell sprouting in vitro. Hence these data support the hypothesis that miR-1307-5p is at least partly responsible for impaired vasculature in tumors overexpressing pre-miR-1307.
Project description:Despite the advances in the understanding of Huntington’s disease (HD), there is the need for molecular biomarkers to categorize mutation-carriers during the preclinical stage of the disease preceding functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are downregulated early in mutation-carriers and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p, and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR-21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species highlighting the progression and cognitive changes occurring at the premanifest stage.
Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.
Project description:Chronic pelvic pain syndrome (CPPS) and chronic prostatitis (CP) is difficult to distinguish from each other, herein termed CP/CPPS. This study aimed at gaining further insight into the change of extracellular vesicles (EVs) in prostatic fluid of male CPPS. From December 2019 to November 2020, after clinical screening, 24 patients with CPPS without obvious urinary symptoms and 13 healthy male participants were included. EVs were isolated from expressed prostatic secretion (EPS) of all subjects. The small non-coding ribonucleic acid (sncRNA) expression of EVs was sequenced, analysed, and validated by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The results showed that plenty of sncRNAs were differentially expressed between the patients and healthy participants. Further qRT–PCR assays validated that several chronic pain-related miRNAs, including miR-204-5p, let-7d-3p, let-7b-3p, let-7c-3p, miR-146a-5p, and miR-320a-5p, were differentially expressed. Series sncRNAs including several chronic pain-related miRNAs were altered in EVs in prostatic fluid of patients with CPPS, which may serve as diagnostic markers for CPPS.