Perfil de metilação global de DNA em células MCF-7 e MCF-10A após exposição transiente de nanopartículas de maghemita funcionalizadas com citrato

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Pós-Graduação em Nanociências e Nanobiotecnologia, 2014.Introdução: Diversos estudos reportam alterações na expressão gênica em resposta àexposição de células à nanomateriais, mas até o presente momento não há nenhumestudo sobre a toxicidade a nível epigenético causada por nanoestruturas e seus efeitosem sucessivas gerações celulares. Portanto, torna-se necessário estudar esses fenômenosa fim de contribuir no desenvolvimento de nanopartículas mais adequadas paraaplicações biológicas. Objetivo: Avaliar o perfil de metilação global de DNA emcélulas MCF-7 e MCF-10A em cultivo após a cessão da exposição de nanopartículas demaghemita funcionalizadas com citrato. Materiais e métodos: As NPM-citrato foramsintetizadas pelo método de coprecipitação de Fe (II) e Fe (III) e adição direta de ácidocítrico. As caracterizações das NPM-citrato foram realizadas por microscopia (MET,HRTEM, MEV e AFM) e por diâmetro hidrodinâmico e potencial zeta. Para detectar asconcentrações sub-letais IC-10 e IC-20, foram realizados a exclusão de viabilidade porcontagem de células coradas com Azul Tripan e o ensaio de citotoxicidade peladetecção da Lactato Desidrogenase (LDH). O ensaio de proliferação celular foirealizado no sistema xCELLigence™ (Roche/ACEA). A detecção de ferro intracelularfoi realizada pelo ensaio do Azul da Prússia. O perfil de metilação global de DNA foirealizado por ensaio colorimétrico. A expressão das DNMTs foi realizada por qRTPCR.Resultados: As NPM-citrato causaram efeito citostático em células MCF-7 eMCF-10A quando administradas nas concentrações 30 e 60μgFe/mL durante 24h. Apósa cessão da exposição das NPM-citrato, verificou-se que a proliferação das célulasMCF-7 tratadas foi maior que das células não tratadas. Além disso, foi constatado queas NPM-citrato encontravam-se no interior das células durante todo o experimento e quehavia uma dinâmica de metilação de DNA, mesmo após a exposição transiente dasNPM-citrato. Também foram identificadas diferenças entre o acúmulo de transcritos deDNA metiltransferases. Discussão: Os nanomateriais possuem um risco intrínseco emaplicações biológicas, mesmo quando administrados em concentrações consideradasnão-tóxicas por meio de técnicas convencionais. Isso porque seus efeitos em sistemasbiológicos podem se estender a múltiplas gerações, mesmo durante exposiçãotransiente. Conclusão: As NPM-citrato promovem alterações significativas no perfil demetilação global de DNA em células MCF-7 e não promovem em MCF-10A e essefenômeno pode ser explorado para aplicações biomédicas futuras. _________________________________________________________________________________ ABSTRACTIntroduction: Several studies have reported changes in gene expression in response toexposure of cells to nanomaterials, but to date there is no study on the toxicity causedby nanostructures at epigenetic level and their effects in successive cell generations.Therefore, it becomes necessary to study these phenomena in order to contribute to thedevelopment of more appropriate nanoparticles for biological applications. Objective:To evaluate the profile of global DNA methylation in MCF-7 and MCF-10A cells inculture after cessation of exposure to maghemite nanoparticles functionalized with citricacid. Methods: The NPM-citrate were synthesized by coprecipitation of Fe (II) and Fe(III) method and direct addition of citric acid. The characterizations of NPM-citratewere performed by microscopy (TEM, HRTEM, SEM and AFM) and by analysis of thehydrodynamic diameter and zeta potential. To detect the sub-lethal concentrations IC-10and IC-20, we performed the counting of the cells stained with Trypan Blue andcytotoxicity assay for detection of lactate dehydrogenase (LDH). The cell proliferationassay was performed in xCELLigence ™ (Roche / ACEA) system. Detection ofintracellular iron assay was performed by Prussian Blue. The profile global DNAmethylation was performed by colorimetric assay. The expression of DNMTs wasperformed by qRT-PCR. Results: NPM-citrate caused cytostatic effect on MCF-7 andMCF-10A cells when given at concentrations of 30 and 60μgFe/ml for 24h. After thetransfer of the NPM-citrate exposure, it was found that the proliferation of MCF-7treated cells was higher than untreated cells. Furthermore, it was found that the NPMcitrateis found inside the cells throughout the experiment and had a dynamic DNAmethylation even after transient exposure of NPM-citrate. Differences between thetranscript accumulation of DNA methyltransferases were also identified. Discussion:The combination of nanotechnology and epigenetics is still poorly understood becausethese are frontier areas of knowledge. Thus, nanomaterials have an intrinsic risk inbiological applications, even when administered in non-toxic concentrations consideredby conventional techniques. This is because their effects on biological systems can beextended to multiple generations, even during transient exposure. Conclusion: TheNPM-citrate promote significant changes in global DNA methylation in MCF-7 cellsand do not promote in MCF-10A and this phenomenon can be exploited for futurebiomedical applications

A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells

Background: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs. Methods: We investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors. Results: The present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression. Conclusion: Our study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro

Exploring the role of non-coding RNAs in ALS: from disease onset to possible applications to counteract muscle degeneration

Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease whose causes remain largely unknown and whose therapeutic options are ineffective in the long term. Several studies suggest that non-coding RNAs and transcription factors, important regulators of gene expression, are critical for ALS onset and progression. Increasing evidence suggests that molecular alterations in muscle fibers in the presymptomatic phase can trigger ALS pathology. However, there is still a huge gap in the knowledge about the molecular mechanisms that compromise the muscle synapses and the consequences during the regeneration process. In the present study, we investigated the role of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and transcription factors (TFs) in the regulation of muscle functioning and plasticity. First, we analyzed the non-coding and coding differentially expressed genes of skeletal muscle from SOD1*G93A mice, an established model for ALS studies. We identified 3 miRNAs (miR-152, miR-193a, and miR-193b) that were predicted to target genes that encode proteins with key functions in the maintenance of neuromuscular junction (NMJ). Our analysis revealed an aberrant expression of genes that encodes for acetylcholine receptors (AChRs) subunits and the results strongly suggest that the 3 miRNAs are involved in the adult-to-fetal (ε to γ) switch of AChRs isoforms in ALS. In vitro assays confirmed that the miRNAs indeed bind the 3’UTR of target genes and effectively induce their degradation. In vivo functional assays corroborate the involvement of miR-152, miR-193a, and miR-193b in the onset of myopathies. The overexpression of the pooled miRNAs in healthy mice induced loss of muscle mass and weight reduction. The mechanisms responsible for muscle atrophy induction were also investigated. High levels of miR-152, miR-193a, and miR-193b induce the impairment of synaptic transmissions and disrupt NMJ functioning. Interestingly, we also observed the fast-to-slow myofiber shift phenomenon as a consequence of the treatment. Altogether, these results suggest that miR-152, miR-193a, and miR-193b could possibly trigger ALS, since their aberrant expression occurs in the early-stage disease. Next, we evaluated the crosstalk between miRNAs-lncRNAs-TFs in muscle functions to reveal novel networks disturbed in myopathies and in muscle development. The transcriptomic analysis associated with specific bioinformatic tools for the predictions of intermolecular interactions allowed us to identify 139 networks that may be involved in the modulation of muscle functions. To select the targets of interest for further characterization, we investigated the expression of a list of those regulatory genes in other muscle atrophy models (denervation and starvation) and during in vitro differentiation. The dynamic expression of the lncRNA Gas5 and the TF Runx1 (possible targets of miR-30) lead us to select them for the next analyses. The in vitro overexpression of three miRNAs from the miR-30 family corroborated our predictions, specifically the upregulation of miR-30 caused the downregulation of Runx1 and the isoforms 5 and 6 of Gas5. Our gain-of-function assays showed that miR-30e promoted myotube fusion, producing thicker myotubes. The transcriptomic analysis of myotubes overexpressing miR-30e revealed its involvement in muscle functions and its ability to negatively regulate the cell cycle. Differently, myoblasts overexpressing Gas5 overexpress genes that promote apoptosis. We showed that miR-30 and Gas5 behave diametrically opposite with regard to regulation of 6 mitochondrial structure. miR-30 promotes mitochondria network fusion while Gas5 induces mitochondria fragmentation and disruption of mitochondria cristae. These results highlight the importance of non-coding RNAs in the regulation of muscle cell activity also guiding them toward disease development and pave the way for new treatments for ALS and other myopathies.Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease whose causes remain largely unknown and whose therapeutic options are ineffective in the long term. Several studies suggest that non-coding RNAs and transcription factors, important regulators of gene expression, are critical for ALS onset and progression. Increasing evidence suggests that molecular alterations in muscle fibers in the presymptomatic phase can trigger ALS pathology. However, there is still a huge gap in the knowledge about the molecular mechanisms that compromise the muscle synapses and the consequences during the regeneration process. In the present study, we investigated the role of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and transcription factors (TFs) in the regulation of muscle functioning and plasticity. First, we analyzed the non-coding and coding differentially expressed genes of skeletal muscle from SOD1*G93A mice, an established model for ALS studies. We identified 3 miRNAs (miR-152, miR-193a, and miR-193b) that were predicted to target genes that encode proteins with key functions in the maintenance of neuromuscular junction (NMJ). Our analysis revealed an aberrant expression of genes that encodes for acetylcholine receptors (AChRs) subunits and the results strongly suggest that the 3 miRNAs are involved in the adult-to-fetal (ε to γ) switch of AChRs isoforms in ALS. In vitro assays confirmed that the miRNAs indeed bind the 3’UTR of target genes and effectively induce their degradation. In vivo functional assays corroborate the involvement of miR-152, miR-193a, and miR-193b in the onset of myopathies. The overexpression of the pooled miRNAs in healthy mice induced loss of muscle mass and weight reduction. The mechanisms responsible for muscle atrophy induction were also investigated. High levels of miR-152, miR-193a, and miR-193b induce the impairment of synaptic transmissions and disrupt NMJ functioning. Interestingly, we also observed the fast-to-slow myofiber shift phenomenon as a consequence of the treatment. Altogether, these results suggest that miR-152, miR-193a, and miR-193b could possibly trigger ALS, since their aberrant expression occurs in the early-stage disease. Next, we evaluated the crosstalk between miRNAs-lncRNAs-TFs in muscle functions to reveal novel networks disturbed in myopathies and in muscle development. The transcriptomic analysis associated with specific bioinformatic tools for the predictions of intermolecular interactions allowed us to identify 139 networks that may be involved in the modulation of muscle functions. To select the targets of interest for further characterization, we investigated the expression of a list of those regulatory genes in other muscle atrophy models (denervation and starvation) and during in vitro differentiation. The dynamic expression of the lncRNA Gas5 and the TF Runx1 (possible targets of miR-30) lead us to select them for the next analyses. The in vitro overexpression of three miRNAs from the miR-30 family corroborated our predictions, specifically the upregulation of miR-30 caused the downregulation of Runx1 and the isoforms 5 and 6 of Gas5. Our gain-of-function assays showed that miR-30e promoted myotube fusion, producing thicker myotubes. The transcriptomic analysis of myotubes overexpressing miR-30e revealed its involvement in muscle functions and its ability to negatively regulate the cell cycle. Differently, myoblasts overexpressing Gas5 overexpress genes that promote apoptosis. We showed that miR-30 and Gas5 behave diametrically opposite with regard to regulation of 6 mitochondrial structure. miR-30 promotes mitochondria network fusion while Gas5 induces mitochondria fragmentation and disruption of mitochondria cristae. These results highlight the importance of non-coding RNAs in the regulation of muscle cell activity also guiding them toward disease development and pave the way for new treatments for ALS and other myopathies

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts

Perturbations of the Proteome and of Secreted Metabolites in Primary Astrocytes from the hSOD1(G93A) ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins—and of secreted metabolites—involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome–lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte–MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis

Secreted Metabolome of ALS-Related hSOD1(G93A) Primary Cultures of Myocytes and Implications for Myogenesis

Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease associated with progressive muscle atrophy, paralysis, and eventually death. Growing evidence demonstrates that the pathological process leading to ALS is the result of multiple altered mechanisms occurring not only in MNs but also in other cell types inside and outside the central nervous system. In this context, the involvement of skeletal muscle has been the subject of a few studies on patients and ALS animal models. In this work, by using primary myocytes derived from the ALS transgenic hSOD1(G93A) mouse model, we observed that the myogenic capability of such cells was defective compared to cells derived from control mice expressing the nonpathogenic hSOD1(WT) isoform. The correct in vitro myogenesis of hSOD1(G93A) primary skeletal muscle cells was rescued by the addition of a conditioned medium from healthy hSOD1(WT) myocytes, suggesting the existence of an in trans activity of secreted factors. To define a dataset of molecules participating in such safeguard action, we conducted comparative metabolomic profiling of a culture medium collected from hSOD1(G93A) and hSOD1(WT) primary myocytes and report here an altered secretion of amino acids and lipid-based signaling molecules. These findings support the urgency of better understanding the role of the skeletal muscle secretome in the regulation of the myogenic program and mechanisms of ALS pathogenesis and progression

Melittin sensitizes skin squamous carcinoma cells to 5-fluorouracil by affecting cell proliferation and survival

Combined 5-fluorouracil (5-FU) and melittin (MEL) is believed to enhance cytotoxic effects on skin squamous cell carcinoma (SCC). However, the rationale underlying cytotoxicity is fundamentally important for a proper design of combination chemotherapy, and to provide translational insights for future therapeutics in the dermatology field. The aim was to elucidate the effects of 5-FU/MEL combination on the viability, proliferation and key structures of human squamous cell carcinoma (A431). Morphology, plasma membrane, DNA, mitochondria, oxidative stress, cell viability, proliferation and cell death pathways were targeted for investigation by microscopy, MTT, trypan blue assay, flow cytometry and real-time cell analysis. 5-FU/MEL (0.25 µM/0.52 µM) enhanced the cytotoxic effect in A431 cells (74.46%, p < .001) after 72 h exposure, showing greater cytotoxic effect when compared to each isolated compound (45.55% 5-FU and 61.78% MEL). The results suggest that MEL induces plasma membrane alterations that culminate in a loss of integrity at subsequent times, sensitizing the cell to 5-FU action. DNA fragmentation, S and G2/M arrest, disruption of mitochondrial metabolism, and alterations in cell morphology culminated in proliferation blockage and apoptosis. 5-FU/MEL combination design optimizes the cytotoxic effects of each drug at lower concentrations, which may represent an innovative strategy for SCC therapy

Insights into how environment shapes post\u2010mortem RNA transcription in mouse brain

Most biological features that occur on the body after death were already deciphered by traditional medicine. However, the molecular mechanisms triggered in the cellular microenvironment are not fully comprehended yet. Previous studies reported gene expression alterations in the post\u2010mortem condition, but little is known about how the environment could in uence RNA degradation and transcriptional regulation. In this work, we analysed the transcriptome of mouse brain after death under three concealment simulations (air exposed, buried, and submerged). Our analyses identi ed 2,103 genes di erentially expressed in all tested groups 48 h after death. Moreover, we identi ed 111 commonly upregulated and 497 commonly downregulated genes in mice from the concealment simulations. The gene functions shared by the individuals from the tested environments were associated with RNA homeostasis, in ammation, developmental processes, cell communication, cell proliferation, and lipid metabolism. Regarding the altered biological processes, we identi ed that the macroautophagy process was enriched in the upregulated genes and lipid metabolism was enriched in the downregulated genes. On the other hand, we also described a list of biomarkers associated with the submerged and buried groups, indicating that these environments can in uence the post\u2010mortem RNA abundance in its particular way

Liposomal paclitaxel induces apoptosis, cell death, inhibition of migration capacity and antitumoral activity in ovarian cancer

The main goal of this study is to evaluate the efficacy of the paclitaxel (PTX) drug formulated with a liposomal nanosystem (L-PTX) in a peritoneal carcinomatosis derived from ovarian cancer. In vitro cell viability studies with the human ovarian cancer line A2780 showed a 50% decrease in the inhibitory concentration for L-PTX compared to free PTX. A2780 cells treated with the L-PTX formulation demonstrated a reduced capacity to form colonies in comparison to those treated with PTX. Cell death following L-PTX administration hinted at apoptosis, with most cells undergoing initial apoptosis. A2780 cells exhibited an inhibitory migration profile when analyzed by Wound Healing and real-time cell analysis (xCELLigence) methods after L-PTX administration. This inhibition was related to decreased expression of the zinc finger E-box-binding homeobox 1 (ZEB1) and transforming growth factor 2 (TGF-β2) genes. In vivo L-PTX administration strongly inhibited tumor cell proliferation in ovarian peritoneal carcinomatosis derived from ovarian cancer, indicating higher antitumor activity than PTX. L-PTX formulation did not show toxicity in the mice model. This study demonstrated that liposomal paclitaxel formulations are less toxic to normal tissues than free paclitaxel and are more effective in inhibiting tumor cell proliferation/migration and inducing ZEB1/TGF-β2 gene expression