Browsing by Subject "microRNA"

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  • Rekker, Kadri; Altmae, Signe; Suhorutshenko, Marina; Peters, Maire; Martinez-Blanch, Juan F.; Codoner, Francisco M.; Vilella, Felipe; Simon, Carlos; Salumets, Andres; Velthut-Meikas, Agne (2018)
    The endometrium undergoes extensive changes to prepare for embryo implantation and microRNAs (miRNAs) have been described as playing a significant role in the regulation of endometrial receptivity. However, there is no consensus about the miRNAs involved in mid-secretory endometrial functions. We analysed the complete endometrial miRNome from early secretory (pre-receptive) and mid-secretory (receptive) phases from fertile women and from patients with recurrent implantation failure (RIF) to reveal differentially expressed (DE) miRNAs in the mid-secretory endometrium. Furthermore, we investigated whether the overall changes during early to mid-secretory phase transition and with RIF condition could be reflected in blood miRNA profiles. In total, 116 endometrial and 114 matched blood samples collected from two different population cohorts were subjected to small RNA sequencing. Among fertile women, 91 DE miRNAs were identified in the mid-secretory vs. early secretory endometrium, while no differences were found in the corresponding blood samples. The comparison of mid-secretory phase samples between fertile and infertile women revealed 21 DE miRNAs from the endometrium and one from blood samples. Among discovered novel miRNAs, chr2_4401 was validated and showed up-regulation in the mid-secretory endometrium. Besides novel findings, we confirmed the involvement of miR-30 and miR-200 family members in mid-secretory endometrial functions.
  • Hänninen, Mikko; Jäntti, Toni; Tolppanen, Heli; Segersvärd, Heli; Tarvasmäki, Tuukka; Lassus, Johan; Vausort, Melanie; Devaux, Yvan; Sionis, Alessandro; Tikkanen, Ilkka; Harjola, Veli-Pekka; Lakkisto, Päivi (2020)
    Cardiogenic shock (CS) is a life-threatening emergency. New biomarkers are needed in order to detect patients at greater risk of adverse outcome. Our aim was to assess the characteristics of miR-21-5p, miR-122-5p, and miR-320a-3p in CS and evaluate the value of their expression levels in risk prediction. Circulating levels of miR-21-5p, miR-122-5p, and miR-320a-3p were measured from serial plasma samples of 179 patients during the first 5-10 days after detection of CS, derived from the CardShock study. Acute coronary syndrome was the most common cause (80%) of CS. Baseline (0 h) levels of miR-21-5p, miR-122-5p, and miR-320a-3p were all significantly elevated in nonsurvivors compared to survivors (p <0.05 for all). Above median levels at 0h of each miRNA were each significantly associated with higher lactate and alanine aminotransferase levels and decreased glomerular filtration rates. After adjusting the multivariate regression analysis with established CS risk factors, miR-21-5p and miR-320a-3p levels above median at 0 h were independently associated with 90-day all-cause mortality (adjusted hazard ratio 1.8 (95% confidence interval 1.1-3.0), p = 0.018; adjusted hazard ratio 1.9 (95% confidence interval 1.2-3.2), p = 0.009, respectively). In conclusion, circulating plasma levels of miR-21-5p, miR-122-5p, and miR-320a-3p at baseline were all elevated in nonsurvivors of CS and associated with markers of hypoperfusion. Above median levels of miR-21-5p and miR-320a-3p at baseline appear to independently predict 90-day all-cause mortality. This indicates the potential of miRNAs as biomarkers for risk assessment in cardiogenic shock.
  • Jäntti, Toni; Segersvärd, Heli; Tolppanen, Heli; Tarvasmäki, Tuukka; Lassus, Johan; Devaux, Yvan; Vausort, Melanie; Pulkki, Kari; Sionis, Alessandro; Bayes-Genis, Antoni; Tikkanen, Ilkka; Lakkisto, Päivi; Harjola, Veli-Pekka (2019)
    Aims The role of microRNAs has not been studied in cardiogenic shock. We examined the potential role of miR-423-5p level to predict mortality and associations of miR-423-5p with prognostic markers in cardiogenic shock. Methods and results We conducted a prospective multinational observational study enrolling consecutive cardiogenic shock patients. Blood samples were available for 179 patients at baseline to determine levels of miR-423-5p and other biomarkers. Patients were treated according to local practice. Main outcome was 90 day all-cause mortality. Median miR-423-5p level was significantly higher in 90 day non-survivors [median 0.008 arbitrary units (AU) (interquartile range 0.003-0.017) vs. 0.004 AU (0.002-0.009), P = 0.003]. miR-423-5p level above median was associated with higher lactate (median 3.7 vs. 2.4 mmol/L, P = 0.001) and alanine aminotransferase levels (median 68 vs. 35 IU/L, P <0.001) as well as lower cardiac index (1.8 vs. 2.4, P = 0.04) and estimated glomerular filtration rate (56 vs. 70 mL/min/1.73 m(2), P = 0.002). In Cox regression analysis, miR-423-5p level above median was associated with 90 day all-cause mortality independently of established risk factors of cardiogenic shock [adjusted hazard ratio 1.9 (95% confidence interval 1.2-3.2), P = 0.01]. Conclusions In cardiogenic shock patients, above median level of miR-423-5p at baseline is associated with markers of hypoperfusion and seems to independently predict 90 day all-cause mortality.
  • Parkkinen, Ilmari (Helsingfors universitet, 2018)
    MicroRNAs are ~22 nucleotide long RNA strands which regulate gene expression by binding to the 3’UTRs of messenger RNAs. MicroRNAs are predicted to regulate about a half of all protein-coding genes in the human genome thus affecting many cellular processes. One crucial part of microRNA biogenesis is the cleaving of pre-miRNA strands into mature microRNAs by the type III RNase enzyme, Dicer. Dicer has been shown to be downregulated due to aging and in many disease states. Particularly central nervous system disorders are linked to dysregulated microRNA processing. According to the latest studies, Dicer is crucial to the survival of dopaminergic neurons and conditional Dicer knockout mice show severe nigrostriatal dopaminergic cell loss, which is a hallmark of Parkinson’s disease. By activating Dicer with a small-molecule drug, enoxacin, the survival of dopaminergic cells exposed to stress is significantly improved. However, enoxacin, which is a fluoroquinolone antibiotic, activates Dicer only at high concentrations (10-100 μM) and is polypharmacological, which may cause detrimental side effects. Therefore, enoxacin is not a suitable drug candidate for Dicer deficiencies and better Dicer-activating drug candidates are needed. The aim of this work was to develop a cell-based fluorescent assay to screen for Dicer-activating compounds. Assays which measure Dicer activity have already been developed, but they have some pitfalls which don’t make them optimal to use for high-throughput screening of Dicer-activating compounds. Some are cell-free enzyme-based assays and thus neglect Dicer in its native context. The RNA to be processed by Dicer does not represent a common mammalian RNA type. Most assays do not have internal normalizing factors, such as a second reporter protein to account for e.g. cell death, or the analysis method is not feasible for high-throughput screening data. Considering these disadvantages, the study started by designing a reporter plasmid in silico. The plasmid expresses two fluorescent proteins, mCherry (red) and EGFP (green), and a mCherry transcripttargeting siRNA implemented into a pre-miR155 backbone which is processed by Dicer. Thus, measuring the ratios of red and green fluorescence intensities will give an indication on Dicer activity. The plasmid also has additional regulatory elements for stabilizing expression levels. The plasmid was then produced by molecular cloning methods and its functionality was tested with Dicer-modulating compounds. The assay was optimised by testing it in different cell lines and varying assay parameters, and stable cell lines were created to make large-scale screening more convenient. Finally, a small-scale screen was done with ten pharmacologically active compounds. Transiently transfected, in Chinese hamster ovarian cells, mCherry silencing was too efficient for reliable detection of improvement in silencing efficiency due to floor effect. With an inducible, Tet-On, system in FLP-IN 293 T-Rex cells, the expression could be controlled by administering doxycycline and the improvement in silencing was quantifiable. The assay seemed to be functional after 72 hours and 120 hours of incubation using enoxacin (100 μM) as a positive control. However, the screening found no compounds to significantly reduce mCherry/EGFP fluorescence ratio and, additionally, the effect of enoxacin was abolished. Therefore, a more thorough analysis on the effects of enoxacin was done and, although statistically significant, enoxacin was only marginally effective in reducing mCherry/EGFP fluorescence ratio after 72 hours of treatment. It should be noted from the small-scale screening that metformin and BDNF, compounds previously shown to elevate Dicer levels, showed similar effects to enoxacin. The quality of the assay in terms of high-throughput screening was determined by calculating Zfactors and coefficients of variations for the experiments, which showed that the variability of the assay was acceptable, but the differences between controls was not large enough for reliable screening. In conclusion, the effects of metformin and BDNF should be further studied and regarding the assay, more optimisation is needed for large-scale, high-throughput, screening to be done with minimal resources.
  • Soleimanbeigi, Shirin (Helsingin yliopisto, 2020)
    Selective degeneration and dysregulation of specific neuronal populations is a common hallmark shared by neurodegenerative diseases affecting the aging population. Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases with debilitating clinical manifestations that follow a chronic and progressive course. Pathological hallmarks of PD involve gradual and specific loss of DA (DA) neurons and widespread presence of Lewy body (LB) inclusions that consist of aggregated presynaptic protein, α-Synuclein (αSyn). Treatment of PD remains to be at symptomatic management as the underlying mechanisms that trigger neurodegeneration are still not fully elucidated. Over the past two decades, microRNAs (miRNAs) have become a major area of interest within biomedical fields and gained increasing momentum in the context of neurodegenerative diseases. In recent developments, changes in mature miRNA profiles have been reported in aging tissue and many age-related diseases, including PD. More recently, a number of studies have found that the most essential enzyme in the miRNA biogenesis pathway, Dicer, exhibits reduced expression with aging. To these ends, a genetic mouse model based on heterozygous knockout of Dicer (DicerHET) was introduced to simulate Dicer downregulation. Initial investigations identified the DicerHET model as a promising tool for studying the relationship between disrupted miRNA biogenesis and neurodegeneration associated with PD. To facilitate future investigations and speed up screening of potential therapeutic compounds using this genetic model, in the current work, we aimed to produce a DicerHET in vitro model with a practical and convenient genetic engineering approach. The main focus of this work was to validate the model and establish a standardized reproducible approach suitable for research that addresses the role of miRNA biogenesis in PD. The desired DicerHET genotype was generated in vitro by employing traditional Cre/loxP system in conjunction with a virally mediated Cre expression. More specifically, primary cortical cultures, derived from Dicer flox/+ mice embryos, were transduced with Cre expressing lentiviral vectors (lenti-hSYN-T2A-Cre) to delete the “floxed” Dicer allele. To establish optimal parameters for the procedure, we analysed recombination efficiency under different transduction conditions. The most efficient recombination was achieved after 5 days of induction in cultures. However, we observed that DicerHET genotype did not attenuate survival of the cells, as assessed by immunohistochemistry. Further, as a proof of concept, we exposed the DicerHET cultures to pre-formed fibrils (PFFs) - a PD related stressor that causes αSyn aggregation. pSer129-αSyn-positive LB-like aggregates were detected in all the PFF-treated cultures, however, with a greater accumulation in the DicerHET cultures. Interestingly, increased aggregation was not accompanied by increased cell death, suggesting that DicerHET genotype does not increase vulnerability of cortical neurons to pSer129-αSyn aggregation. Based on our earlier studies we presume that DA neurons may bear a specific vulnerability towards the age-related Dicer depletion. More conclusive evidence on this intriguing relationship could be provided in future research using the DicerHET model that can be readily applied to primary DA cultures.
  • Rekker, Kadri; Tasa, Tonis; Saare, Merli; Samuel, Kulli; Kadastik, Ulle; Karro, Helle; Goette, Martin; Salumets, Andres; Peters, Maire (2018)
    microRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.
  • Kilpinen, Lotta; Parmar, Amarjit; Greco, Dario; Korhonen, Matti; Lehenkari, Petri; Saavalainen, Paivi; Laitinen, Saara (2016)
    Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together.
  • Varendi, Kärt; Mätlik, Kert; Andressoo, Jaan-Olle (2015)
    During the past decade, the identification of microRNA (miR) targets has become common laboratory practice, and various strategies are now used to detect interactions between miRs and their mRNA targets. However, the current lack of a standardized identification process often leads to incomplete and/or conflicting results. Here, we review the problems most commonly encountered when verifying miR-mRNA interactions, and we propose a workflow for future studies. To illustrate the challenges faced when validating a miR target, we discuss studies in which the regulation of brain-derived neurotrophic factor by miRs was investigated, and we highlight several controversies that emerged from these studies. Finally, we discuss the therapeutic use of miR inhibitors, and we discuss several questions that should be addressed before proceeding to preclinical testing.
  • Konovalova, Julia; Gerasymchuk, Dmytro; Parkkinen, Ilmari; Chmielarz, Piotr; Domanskyi, Andrii (2019)
    MicroRNAs are post-transcriptional regulators of gene expression, crucial for neuronal differentiation, survival, and activity. Age-related dysregulation of microRNA biogenesis increases neuronal vulnerability to cellular stress and may contribute to the development and progression of neurodegenerative diseases. All major neurodegenerative disorders are also associated with oxidative stress, which is widely recognized as a potential target for protective therapies. Albeit often considered separately, microRNA networks and oxidative stress are inextricably entwined in neurodegenerative processes. Oxidative stress affects expression levels of multiple microRNAs and, conversely, microRNAs regulate many genes involved in an oxidative stress response. Both oxidative stress and microRNA regulatory networks also influence other processes linked to neurodegeneration, such as mitochondrial dysfunction, deregulation of proteostasis, and increased neuroinflammation, which ultimately lead to neuronal death. Modulating the levels of a relatively small number of microRNAs may therefore alleviate pathological oxidative damage and have neuroprotective activity. Here, we review the role of individual microRNAs in oxidative stress and related pathways in four neurodegenerative conditions: Alzheimer's (AD), Parkinson's (PD), Huntington's (HD) disease, and amyotrophic lateral sclerosis (ALS). We also discuss the problems associated with the use of oversimplified cellular models and highlight perspectives of studying microRNA regulation and oxidative stress in human stem cell-derived neurons.
  • Yoo, Bo Ram (Helsingfors universitet, 2017)
    Cardiovascular disease is one of the leading causes of mortality worldwide. Upon myocardial infarction, billions of cardiomyocytes are lost, a fibrotic scar forms, and the heart's contractile function is compromised. Mammalian cardiomyocytes lose most of their proliferative capacity shortly after birth. This decline in proliferative capacity is associated with a switch from glycolysis to oxidative phosphorylation, yielding more ATP, but also inevitably forming reactive oxygen species (ROS). Therefore, finding a way to extend the proliferative window seems crucial to cardiac repair. microRNAs (miRNAs) are short, single-stranded noncoding RNAs that repress gene expression after transcription by binding to their target mRNAs. SIRT1-7, mammalian homologs of the Sirt2 protein in yeast, have been implicated in the regulation of metabolic homeostasis, cell proliferation, cardiac hypertrophy, and aging. The objective of our research was to investigate the differential expression of SIRT1-7 between day 1 and day 7 neonatal mice. Since cells continue to divide until day 7, we wanted to compare the differences in sirtuin expression during the two time points. By doing so, we hoped to gain insight into ways we could regulate sirtuin protein expression by manipulating miRNA and sirtuin gene expression in diseased hearts, thereby promoting the fetal gene program and inducing cells to reenter the cell cycle. Proteins were isolated from whole cell lysates of cardiac tissue of day 1 and day 7 neonatal mice, and western blotting technique was used to analyze SIRT1-7 expression. Expression of SIRT3 and 7 was significantly higher in day 7 as opposed to day 1 in at least two of the three runs, with SIRT7 levels being higher in day 7 in all three runs. Our study provides a basis for carrying out more quantitative analysis to validate gene and protein expression and protein activity, since expression is different at the gene and protein levels and does not necessarily translate into activity.
  • Gerasymchuk, Dmytro; Hubiernatorova, Anastasiia; Domanskyi, Andrii (2020)
    The cytoskeleton is one of the most mobile and complex cell structures. It is involved in cellular transport, cell division, cell shape formation and adaptation in response to extra- and intracellular stimuli, endo- and exocytosis, migration, and invasion. These processes are crucial for normal cellular physiology and are affected in several pathological processes, including neurodegenerative diseases, and cancer. Some proteins, participating in clathrin-mediated endocytosis (CME), play an important role in actin cytoskeleton reorganization, and formation of invadopodia in cancer cells and are also deregulated in neurodegenerative disorders. However, there is still limited information about the factors contributing to the regulation of their expression. MicroRNAs are potent negative regulators of gene expression mediating crosstalk between different cellular pathways in cellular homeostasis and stress responses. These molecules regulate numerous genes involved in neuronal differentiation, plasticity, and degeneration. Growing evidence suggests the role of microRNAs in the regulation of endocytosis, cell motility, and invasiveness. By modulating the levels of such microRNAs, it may be possible to interfere with CME or other processes to normalize their function. In malignancy, the role of microRNAs is undoubtful, and therefore changing their levels can attenuate the carcinogenic process. Here we review the current advances in our understanding of microRNAs regulating actin cytoskeleton dynamics, CME and cell motility with a special focus on neurodegenerative diseases, and cancer. We investigate whether current literature provides an evidence that microRNA-mediated regulation of essential cellular processes, such as CME and cell motility, is conserved in neurons, and cancer cells. We argue that more research effort should be addressed to study the neuron-specific functions on microRNAs. Disease-associated microRNAs affecting essential cellular processes deserve special attention both from the view of fundamental science and as future neurorestorative or anti-cancer therapies.
  • Rantanen, Mimi (Helsingfors universitet, 2016)
    The main purpose of this study was to participate in setting up the In situ hybridization for microRNA detection at the Department of Pharmacology. In situ hybridization (ISH) is an effective method for detection of molecules like DNA and RNA from paraffin fixed tissue sections. ISH provides information of expression and location of selected target molecules. Challenges of ISH for miRNA are the small size of the miRNAs and requirement of RNase free environment to prevent contamination. Micro-RNAs are single-stranded, noncoding, 19 to 25 nucleotides long RNAs, involved in post-transcriptional gene silencing. MiRNA-1 is known to be upregulated in ischemic heart muscle, which increases arrhythmias and apoptosis in heart. Adminstration of isoprenaline to Wistar rats induces similar conditions as acute myocardial infarction, which leads to rise in mir-1 levels. By in situ hybridization we were able to detect mir-1 from heart tissue and adjust suitable conditions for ISH.
  • Loppi, Sanna; Korhonen, Paula; Bouvy-Liivrand, Maria; Caligola, Simone; Turunen, Tiia A.; Turunen, Mikko P.; de Sande, Ana Hernandez; Kolosowska, Natalia; Scoyni, Flavia; Rosell, Anna; Garcia-Berrocoso, Teresa; Lemarchant, Sighild; Dhungana, Hiramani; Montaner, Joan; Koistinaho, Jari; Kanninen, Katja M.; Kaikkonen, Minna U.; Giugno, Rosalba; Heinäniemi, Merja; Malm, Tarja (2021)
    Ischemic stroke, the third leading cause of death in the Western world, affects mainly the elderly and is strongly associated with comorbid conditions such as atherosclerosis or diabetes, which are pathologically characterized by increased inflammation and are known to influence the outcome of stroke. Stroke incidence peaks during influenza seasons, and patients suffering from infections such as pneumonia prior to stroke exhibit a worse stroke outcome. Earlier studies have shown that comorbidities aggravate the outcome of stroke, yet the mediators of this phenomenon remain obscure. Here, we show that acute peripheral inflammation aggravates stroke-induced neuronal damage and motor deficits specifically in aged mice. This is associated with increased levels of plasma proinflammatory cytokines, rather than with an increase of inflammatory mediators in the affected brain parenchyma. Nascent transcriptomics data with mature microRNA sequencing were used to identify the neuron-specific miRNome, in order to decipher dysregulated miRNAs in the brains of aged animals with stroke and co-existing inflammation. We pinpoint a previously uninvestigated miRNA in the brain, miR-127, that is highly neuronal, to be associated with increased cell death in the aged, LPS-injected ischemic mice. Target prediction tools indicate that miR-127 interacts with several basally expressed neuronal genes, and of these we verify miR-127 binding to Psmd3. Finally, we report reduced expression of miR-127 in human stroke brains. Our results underline the impact of peripheral inflammation on the outcome of stroke in aged subjects and pinpoint molecular targets for restoring endogenous neuronal capacity to combat ischemic stroke.
  • Iivonen, Anna-Pauliina; Känsäkoski, Johanna; Vaaralahti, Kirsi; Raivio, Taneli (2019)
    In approximately half of congenital hypogonadotropic hypogonadism (cHH) patients, the genetic cause remains unidentified. Since the lack of certain miRNAs in animal models has led to cHH, we sequenced human miRNAs predicted to regulate cHH-related genes (MIR7-3, MIR141, MIR429 and MIR200A-C) in 24 cHH patients with Sanger sequencing. A heterozygous variant in MIR200A (rs202051309; general population frequency of 0.02) was found in one patient. Our results suggest that mutations in the studied miRNAs are unlikely causes of cHH. However, the complex interplay between miRNAs and their target genes in these diseases requires further investigations.
  • Mitchell, Robert; Mellows, Ben; Sheard, Jonathan; Antonioli, Manuela; Kretz, Oliver; Chambers, David; Zeuner, Marie-Theres; Tomkins, James E; Denecke, Bernd; Musante, Luca; Joch, Barbara; Debacq-Chainiaux, Florence; Holthofer, Harry; Ray, Steve; Huber, Tobias B; Dengjel, Joern; De Coppi, Paolo; Widera, Darius; Patel, Ketan (BioMed Central, 2019)
    Abstract Background The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair. Methods Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source. Results Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome. Conclusions Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.
  • Polini, Beatrice; Carpi, Sara; Doccini, Stefano; Citi, Valentina; Martelli, Alma; Feola, Sara; Santorelli, Filippo Maria; Cerullo, Vincenzo; Romanini, Antonella; Nieri, Paola (2020)
    Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and -5p transfection in cutaneous melanoma cell lines are investigated. Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.
  • Waldenström macroglobulinemia Group (2018)
    Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) is a rare, chronic B-cell lymphoma with high heritability. We conduct a two-stage genome-wide association study of WM/LPL in 530 unrelated cases and 4362 controls of European ancestry and identify two high-risk loci associated with WM/LPL at 6p25.3 (rs116446171, near EXOC2 and IRF4; OR = 21.14, 95% CI: 14.40–31.03, P = 1.36 × 10 −54 ) and 14q32.13 (rs117410836, near TCL1; OR = 4.90, 95% CI: 3.45–6.96, P = 8.75 × 10 −19 ). Both risk alleles are observed at a low frequency among controls (~2–3%) and occur in excess in affected cases within families. In silico data suggest that rs116446171 may have functional importance, and in functional studies, we demonstrate increased reporter transcription and proliferation in cells transduced with the 6p25.3 risk allele. Although further studies are needed to fully elucidate underlying biological mechanisms, together these loci explain 4% of the familial risk and provide insights into genetic susceptibility to this malignancy. © 2018, The Author(s).