RESUMO
microRNAs (miRNAs) associate with Ago proteins to post-transcriptionally silence gene expression by targeting mRNAs. To characterize the modes of miRNA-binding, we developed a novel computational framework, called optiCLIP, which considers the reproducibility of the identified peaks among replicates based on the peak overlap. We identified 98 999 binding sites for mouse and human miRNAs, from eleven Ago2 CLIP-seq datasets. Clustering the binding preferences, we found heterogeneity of the mode of binding for different miRNAs. Finally, we set up a quantitative model, named miRgame, based on an adaptation of the game theory. We have developed a new algorithm to translate the miRgame into a score that corresponds to a miRNA degree of occupancy for each Ago2 peak. The degree of occupancy summarizes the number of miRNA-binding sites and miRNAs targeting each binding site, and binding energy of each miRNA::RNA heteroduplex in each peak. Ago peaks were stratified accordingly to the degree of occupancy. Target repression correlates with higher score of degree of occupancy and number of miRNA-binding sites within each Ago peak. We validated the biological performance of our new method on miR-155-5p. In conclusion, our data demonstrate that miRNA-binding sites within each Ago2 CLIP-seq peak synergistically interplay to enhance target repression.
Assuntos
Proteínas Argonautas/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Teoria dos Jogos , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Algoritmos , Animais , Sítios de Ligação , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Camundongos , Modelos BiológicosRESUMO
Gaucher disease (GD) is caused by biallelic pathogenic variants in the acid ß-glucosidase gene (GBA1), leading to a deficiency in the ß-glucocerebrosidase (GCase) enzyme activity resulting in the intracellular accumulation of sphingolipids. Skeletal alterations are one of the most disabling features in GD patients. Although both defective bone formation and increased bone resorption due to osteoblast and osteoclast dysfunction contribute to GD bone pathology, the molecular bases are not fully understood, and bone disease is not completely resolved with currently available specific therapies. For this reason, using editing technology, our group has developed a reliable, isogenic, and easy-to-handle cellular model of GD monocytes (GBAKO-THP1) to facilitate GD pathophysiology studies and high-throughput drug screenings. In this work, we further characterized the model showing an increase in proinflammatory cytokines (Interleukin-1ß and Tumor Necrosis Factor-α) release and activation of osteoclastogenesis. Furthermore, our data suggest that GD monocytes would display an increased osteoclastogenic potential, independent of their interaction with the GD microenvironment or other GD cells. Both proinflammatory cytokine production and osteoclastogenesis were restored at least, in part, by treating cells with the recombinant human GCase, a substrate synthase inhibitor, a pharmacological chaperone, and an anti-inflammatory compound. Besides confirming that this model would be suitable to perform high-throughput screening of therapeutic molecules that act via different mechanisms and on different phenotypic features, our data provided insights into the pathogenic cascade, leading to osteoclastogenesis exacerbation and its contribution to bone pathology in GD.
Assuntos
Doença de Gaucher , Humanos , Doença de Gaucher/patologia , Osteogênese , Monócitos/patologia , Sistemas CRISPR-Cas , Diferenciação CelularRESUMO
Complex trait genome-wide association studies (GWAS) provide an efficient strategy for evaluating large numbers of common variants in large numbers of individuals and for identifying trait-associated variants. Nevertheless, GWAS often leave much of the trait heritability unexplained. We hypothesized that some of this unexplained heritability might be due to common and rare variants that reside in GWAS identified loci but lack appropriate proxies in modern genotyping arrays. To assess this hypothesis, we re-examined 7 genes (APOE, APOC1, APOC2, SORT1, LDLR, APOB, and PCSK9) in 5 loci associated with low-density lipoprotein cholesterol (LDL-C) in multiple GWAS. For each gene, we first catalogued genetic variation by re-sequencing 256 Sardinian individuals with extreme LDL-C values. Next, we genotyped variants identified by us and by the 1000 Genomes Project (totaling 3,277 SNPs) in 5,524 volunteers. We found that in one locus (PCSK9) the GWAS signal could be explained by a previously described low-frequency variant and that in three loci (PCSK9, APOE, and LDLR) there were additional variants independently associated with LDL-C, including a novel and rare LDLR variant that seems specific to Sardinians. Overall, this more detailed assessment of SNP variation in these loci increased estimates of the heritability of LDL-C accounted for by these genes from 3.1% to 6.5%. All association signals and the heritability estimates were successfully confirmed in a sample of â¼10,000 Finnish and Norwegian individuals. Our results thus suggest that focusing on variants accessible via GWAS can lead to clear underestimates of the trait heritability explained by a set of loci. Further, our results suggest that, as prelude to large-scale sequencing efforts, targeted re-sequencing efforts paired with large-scale genotyping will increase estimates of complex trait heritability explained by known loci.
Assuntos
LDL-Colesterol/genética , Mapeamento Cromossômico , Loci Gênicos/genética , Variação Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Itália , Polimorfismo de Nucleotídeo Único/genética , População Branca/genéticaRESUMO
Obesity is a growing societal scourge. Recent studies have uncovered that paternal excessive weight induced by an unbalanced diet affects the metabolic health of offspring. These reports mainly employed single-generation male exposure. However, the consequences of multigenerational unbalanced diet feeding on the metabolic health of progeny remain largely unknown. Here, we show that maintaining paternal Western diet feeding for five consecutive generations in mice induces an enhancement in fat mass and related metabolic diseases over generations. Strikingly, chow-diet-fed progenies from these multigenerational Western-diet-fed males develop a 'healthy' overweight phenotype characterized by normal glucose metabolism and without fatty liver that persists for four subsequent generations. Mechanistically, sperm RNA microinjection experiments into zygotes suggest that sperm RNAs are sufficient for establishment but not for long-term maintenance of epigenetic inheritance of metabolic pathologies. Progressive and permanent metabolic deregulation induced by successive paternal Western-diet-fed generations may contribute to the worldwide epidemic of metabolic diseases.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Epigênese Genética , Predisposição Genética para Doença/genética , Doenças Metabólicas/genética , Exposição Paterna , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genéticaRESUMO
During the development of atherosclerotic lesion, s-RNYs (small RNAs of about 24/34 nucleotides) are derived by the processing of long Ro-associated non-coding RNAs (RNYs) in macrophages. The levels of serum s-RNYs have been found significantly upregulated in patients with coronary heart disease (CHD) compared to age-matched CHD-free individuals. The present study aimed to examine the predictive value of serum s-RNYs for CHD events in the general male population. Within the frame of nested-case-control study, the GENES study, we measured the absolute expression of a RNY-derived small RNA, the s-RNY1-5p, in the serum of individuals (without CHD at baseline) who encountered a CHD event within 12 years of follow-up (n = 30) (Cases) and compared them to individuals who remained event-free (Controls) (n = 30). The expression of s-RNY1-5p in serum was significantly upregulated in Cases compared to Controls (p = 0.027). The proportion of CHD event-free was significantly higher among individuals with serum s-RNY1-5p below the median value (631 molecules/mL). In a multivariable model adjusted for age, smoking, hypertension, diabetes and dyslipidemia, the risk of CHD events increased more than fourfold in individuals with serum s-RNY1-5p above the median value (HR, 4.36; 95% CI 1.22-15.60). A positive association with CHD events was also observed when considering s-RNY1-5p as a continuous variable (p = 0.022). Based on our results, we conclude that serum s-RNY1-5p is an independent predictor of CHD events in a general male population and might be a relevant biomarker for early detection of cardiovascular diseases.
Assuntos
Doença das Coronárias/epidemiologia , RNA Longo não Codificante/sangue , Idoso , Aterosclerose/complicações , Biomarcadores/sangue , Estudos de Casos e Controles , Doença das Coronárias/sangue , Doença das Coronárias/complicações , Complicações do Diabetes , Humanos , Hipertensão/complicações , Incidência , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , FumarRESUMO
To date, the knowledge of the genetic determinants behind the modulation of hearing ability is relatively limited. To investigate this trait, we performed Genome-Wide Association Study (GWAS) meta-analysis using genotype and audiometric data (hearing thresholds at 0.25, 0.5, 1, 2, 4, and 8 kHz, and pure-tone averages of thresholds at low, medium, and high frequencies) collected in nine cohorts from Europe, South-Eastern USA, Caucasus, and Central Asia, for an overall number of ~9000 subjects. Three hundred seventy-five genes across all nine analyses were tagged by single nucleotide polymorphisms (SNPs) reaching a suggestive p-value (p < 10-5). Amongst these, 15 were successfully replicated using a gene-based approach in the independent Italian Salus in the Apulia cohort (n = 1774) at the nominal significance threshold (p < 0.05). In addition, the expression level of the replicated genes was assessed in published human and mouse inner ear datasets. Considering expression patterns in humans and mice, eleven genes were considered particularly promising candidates for the hearing function: BNIP3L, ELP5, MAP3K20, MATN2, MTMR7, MYO1E, PCNT, R3HDM1, SLC9A9, TGFB2, and YTHDC2. These findings represent a further contribution to our understanding of the genetic basis of hearing function and its related diseases.
Assuntos
Audição/genética , Animais , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Humanos , Itália , Masculino , Camundongos , Limiar SensorialRESUMO
INTRODUCTION: Ex vivo cell expansion under Good Manufacturing Practice (GMP) guidelines can be performed using medium additives containing human growth factors from platelets. These products can differently affect proliferation of adipose mesenchymal stromal stem cells (ASC). Qualification of medium additive performance is required for validation under GMP regulations: assessment of growth factor concentrations is not sufficient to predict the biological activity of the product batch. Proton nuclear magnetic resonance spectrometry (1H-NMR) and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) provide wide molecular characterization of samples. AIMS: We aimed to assess if 1H-NMR and MALDI-TOF MS techniques can be used as quality control test potentially predicting the impact of a medium additive on cell proliferation. METHODS: We tested the impact on ASC growth rate (cell proliferation assessment and cell morphology analysis) of four medium additives, obtained by different methods from human platelet apheresis product. In order to classify each medium additive, we evaluated growth factor concentrations and spectra obtained by 1H-NMR and by MALDI-TOF MS. RESULTS: Medium additive obtained by CaCl2 activation of platelet rich products induced higher proliferation rate vs additive derived from platelet depleted ones. Additives obtained by freeze-and-thaw methods weakly induced ASC proliferation. As expected, principal component analysis of growth factor concentrations did not unravel specific biochemical features characterizing medium additives in relation with their biological activity. Otherwise, while 1H-NMR showed a partial resolution capacity, analysis of MALDI-TOF MS spectra allowed unambiguous distinction between the medium additives we used to differently stimulate cell growth in vitro. DISCUSSION: MALDI-TOF and, despite limitations, 1H-NMR are promising cost effective and reliable quality controls to classify the potential of a medium additive to promote ASC growth. This can represent, after further investigations and appropriate validation, a significant advantage for GMP compliant manufacturing of advanced cell therapy products.
Assuntos
Meios de Cultura , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética , Controle de Qualidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Plaquetas , Cloreto de Cálcio , Proliferação de Células , Células Cultivadas , Meios de Cultura/química , Humanos , Indústria Manufatureira , Metabolômica/métodosRESUMO
Mammalian apurinic/apyrimidinic endonuclease 1 is a DNA repair enzyme involved in genome stability and expression of genes involved in oxidative stress responses, tumor progression and chemoresistance. However, the molecular mechanisms underlying the role of apurinic/apyrimidinic endonuclease 1 in these processes are still unclear. Recent findings point to a novel role of apurinic/apyrimidinic endonuclease 1 in RNA metabolism. Through the characterization of the interactomes of apurinic/apyrimidinic endonuclease 1 with RNA and other proteins, we demonstrate here a role for apurinic/apyrimidinic endonuclease 1 in pri-miRNA processing and stability via association with the DROSHA-processing complex during genotoxic stress. We also show that endonuclease activity of apurinic/apyrimidinic endonuclease 1 is required for the processing of miR-221/222 in regulating expression of the tumor suppressor PTEN. Analysis of a cohort of different cancers supports the relevance of our findings for tumor biology. We also show that apurinic/apyrimidinic endonuclease 1 participates in RNA-interactomes and protein-interactomes involved in cancer development, thus indicating an unsuspected post-transcriptional effect on cancer genes.APE1 plays an important role in the cellular response to oxidative stress, and mutations are linked to tumor progression and chemoresistance. Here, the authors characterize the interactions of APE1 with RNA and demonstrate a role in microRNA processing.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , MicroRNAs/metabolismo , Neoplasias/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/metabolismoRESUMO
Similarity (or conversely distance) measures are at the heart of most bioinformatic applications. When the similarity involves only a small subset of features out of many, global similarity measures may be significantly affected by noise. Selecting only a subset of (putatively relevant) features for comparison is a widespread solution to the problem albeit affected by arbitrariness and manual intervention. The problem is becoming more and more important due to the increasing amount of experimental data available. In recent years measures based on ranking similarities between two datasets have been proposed. Here, we use one of the proposed rank similarity measures, sharing some aspects with the fraction enrichment score used for protein structure prediction and the gene set enrichment analysis, and test its performance in classifying experiments. The discrimination ability of the similarity measures based on the overlap of ranked genes tested here compares well or better with standard measures of similarity. This conclusion supports the use of rank-based proximity measures to gain further insight in dataset comparisons, particularly on expression data obtained by different techonologies (e.g., RNA-seq and microarrays).
Assuntos
Perfilação da Expressão Gênica/classificação , Análise de Sequência com Séries de Oligonucleotídeos/classificação , Proteínas/genética , Algoritmos , Biologia Computacional/métodosRESUMO
PURPOSE: To assess the efficacy and long-term results of Ethibloc treatment in aneurysmal bone cysts (ABC). METHODS: Thirteen patients with ABC were treated with direct percutaneous Ethibloc injection. Four patients had only one injection and the other nine patients from two to four injections. No severe complications were observed; in two patients a local leakage of Ethibloc from the cyst into the soft tissues occurred but it was temporary and the consequent inflammation self-healed without residua and sequelae. Imaging follow-up lasted from 6 to 67 months and included conventional radiology (CR) and magnetic resonance imaging (MRI), both used in the presurgical phase. RESULTS AND CONCLUSIONS: All images demonstrated a remarkable shrinkage of the cystic lesion and bone cortex thickening. In all patients, circumscribed areas of lucency persisted at radiography, corresponding to residual cystic areas without fluid-fluid levels at MRI. Pain, which was present in all the patients before treatment, was relieved within a month. According to our experience, direct percutaneous Ethibloc injection is effective in the treatment of ABC and can be recommended as the first-choice treatment. Due to its higher sensitivity MRI must be included either in the pretreatment phase to study the multilocular structure or in the imaging follow-up to evaluate the efficacy of Ethibloc in persistently non-responsive areas.