1,721,017 research outputs found
Novel trands in microRNA based theranostics
No full textI miRNA sono una classe di RNA non codificanti in grado di regolare l’espressione genica attraverso l’interazione con la regione 3’UTR del trascritto bersaglio, portando all’inibizione della traduzione o alla degradazione del mRNA bersaglio. A causa della loro breve sequenza (19-25 nucleotidi), i miR sono in grado di riconoscere regioni bersaglio in più trascritti, risultando, coinvolti in più di un processo molecolare. Diverse patologie sono state associate alla disregolazione del profilo di espressione di miR, negli anni sono state quindi elaborate diverse strategie terapeutiche allo scopo di ripristinare i livelli fisiologici di miR.
Molecole in grado di mimare la funzione del miR-210 sono state impiegate nella presente tesi allo scopo di ridurre l’espressione del fattore di trascrizione BCL11A, uno dei principali repressori del gene γ-globinico. Lo studio ha permesso di ottenere due risultati chiave: a) miR-210 riconosce una sequenza presente nella regione codificante del trascritto BCL11A, b) l’incremento dei livelli intracellulari di miR-210 sono associati al decremento di trascritto e proteina BCL11A. I dati suggeriscono che l’impiego di molecole in grado di mimare la funzione di miR-210 possono essere impiegate nel trattamento della β-talassemia.
Le molecole in grado di modulare i livelli intracellulari di miR richiedono un veicolo per essere internalizzate dalle cellule. Nella presente tesi è stata valutata una molecola a struttura calixarenica (ML122) e precedentemente impiegata per la trasfezione di DNA. ML122 è stato testato, sia per la veicolazione di molecole elettricamente cariche (antimiRNA e premiRNA), sia per molecole elettricamente neutre (PNA). I dati hanno dimostrato a) un’elevata efficienza di internalizzazione cellulare delle molecole veicolate da ML122, associata a b) evidenti effetti biologici delle molecole veicolate, che una volta internalizzate, sono rilasciate dal complesso formato con ML122 e svolgono il loro ruolo biologico.
Come dimostrato da Chim nel 2008 i miR sono presenti in diversi fluidi biologici, tra cui il plasma, dove risultano particolarmente stabili, rendendoli ottimi marcatori nell’ambito della diagnostica non invasiva. Partendo da questo assunto i cmiRNA sono stati impiegati come marcatori diagnostici in due differenti ambiti.
Nella prima parte i miR sono stati impiegati come marcatori per la diagnosi di carcinoma del colon-retto giungendo alle seguenti conclusioni: a) i miR sono fisiologicamente rilasciati dalle cellule e ogni linea cellulare presenta un proprio profilo di ‘secrezione’ che risulta indipendente dalle concentrazioni intracellulari di miR, b) la comparazione di due diverse metodiche PCR per la quantificazione di miRNA (RTqPCR e ddPCR) ha dimostrato risultati comparabili, nonostante la ddPCR risulti maggiormente performante, specialmente in campioni molto diluiti c) l’analisi di 3 miR selezionati sulla base di analisi riportate in letteratura, in pazienti affetti da CRC e donatori sani ha dimostrato diversi limiti derivanti dall’impiego di un numero limitato di miR suggerendo la necessità di considerare panelli più ampi di miR.
L’analisi di miRNA circolanti è stata inoltre applicata al campo dell’identificazioni di frodi sportive, in particolar modo i miR sono stati proposti come marcatori di autoemotrasfusione. L’analisi di un numero seppur limitato di campioni, con metodica microarray ha permesso di identificare due diverse liste di miR candidati marcatori di autoemotrasfusione. Partendo dall’assunto che durante i 2 processi chiave dell’ABT: prelievo del sangue e reinfusione, generano un’alterazione dei livelli di ossigeno, è stata stilata una prima lista di 8 miR, la cui espressione risulta modulata in risposta alla variazione dei livelli di ossigeno. Traendo vantaggio dai dati di microarray è stata inoltre, proposta una seconda lista di miR, la cui espressione è modulata in seguito alla reinfusione.MiRNAs are a class of small non-coding RNA of about 19-23 nucleotides in length able to act as regulators of gene expression thanks to their ability to bind the 3’UTR which results in inhibition of translation or in mRNA degradation. Due to their short sequence, they can bind more than one transcript, so they may be involved in more than one biological pathway. Since their first identification in 1993, they have been associated to a long list of physiological or pathological conditions. The dysregulation of miRNA profile may be associated to several diseases, so therapies based on the restore of physiological miRNA levels may have huge impact on several pathologies, for this reason molecules able to both increase or decrease miRNA levels have been recently developed.
Through miRNAs levels regulation is possible to indirectly regulate their targets levels. This evidence was investigated in this thesis to reduce levels of BCL11A, one of the principal repressor of γ-globin gene. The following key results: a) miR-210 interaction with BCL11A coding region was demonstrated with SPR-based analysis, b) the increase of miR-210 intracellular levels leads to a decrease of BCL11A transcript and protein, encouraged us to consider the employment of miR-210 mimicking molecules as possible therapeutic approach to reduce BCL11A expression in the field of β-thalassemia treatment.
Modulation of miRNAs levels into cells can be achieve with different kinds of molecules and most of them generally require an appropriate vehicle. At this propose we investigated with encouraging results a calixarene-based structure compound called ML122, previously used for DNA delivery, to vehicle miRNA-based molecules and PNAs. a) High transfection efficiency, associated with b) evident biological effects obtained when both premiRNA molecules and PNAs are vehicled with ML122, allow us to consider ML122 as a possible alternative to commercial available transfection agents.
MiRNAs are present not only into cells but as demonstrated by Chim and colleagues they were found also in several body fluids, including plasma, in which have been demonstrated to be very stable. Several groups employed circulating miRNAs at diagnostic or prognostic propose opening a new important issue in the field of the non-invasive diagnostic techniques.
In the present thesis we employed the non-invasive diagnostic technique in two different fields. First, we considered circulating miRNA in colorectal cancer diagnosis and management. In this section, three important massages were obtained a) miRNA are normally released by cells, the release pattern is different in each cell line and often differs from the miRNA pattern into cells, b) a comparison between two different techniques, RTqPCR and ddPCR, demonstrates how the two techniques gave comparable results, even if ddPCR for technical issue is more suitable for miRNA quantification in plasma samples, c) analysis of the three selected miRNAs in CRC patients and healthy controls demonstrates how additional miRNAs (at 6 or 7 miRNAs) are needed to identify patterns associated to CRC patients.
The analysis of cmiRNAs was also, not applied to a health problem but to identify an illicit practice in sport such as autologous blood transfusion. The analysis of a limited number of samples using a high-throughput miRNA analysis method, i.e. microarray, allow us to identify two different list of miRNAs possible biomarkers for the detection of ABT: (a) a list of 8 miRNAs which modulation may be related to different oxygen availability immediately after the two key steps of ABT practice i.e. blood withdrawal and blood reinfusion. Moreover, a second list (b) was obtained considering all expressed miRNAs in our plasma samples. Despite the number of analysed samples is limited (6 ABT trained subjects and 3 control pools) preliminary encouraging data were obtained, which of course need to be confirmed increasing the samples size
Preliminary results and a theoretical perspective of co‐treatment using a miR‐93‐5p mimic and aged garlic extract to inhibit the expression of the pro‐inflammatory interleukin‐8 gene
No full textThe coronavirus disease-19 (COVID-19) pandemic has been a very significant health issue in the period between 2020 and 2023, forcing research to characterize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and to develop novel therapeutic approaches. Interleukin-6 (IL-6) and IL-8 are considered significant therapeutic targets for COVID-19 and emerging evidence has suggested that microRNAs (miRNAs/miRs) serve a key role in regulating these genes. MiRNAs are short, 19-25 nucleotides in length, non-coding RNAs that regulate gene expression at the post-transcriptional level through the sequence-selective recognition of the 3'-untranslated region (3'-UTR) of the regulated mRNAs, eventually repressing translation, commonly, via mRNA degradation. For example, among several miRNAs involved in the regulation of the COVID-19 'cytokine storm', miR-93-5p can inhibit IL-8 gene expression by directly targeting the 3'-UTR of IL-8 mRNA. In addition, miR-93-5p can regulate Toll-like receptor-4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4) expression, thus affecting the nuclear factor-kappa B (NF-kappa B) pathway and the expression of NF-kappa B-regulated genes, such as IL-6, IL-1 beta and other hyper-expressed genes during the COVID-19 'cytokine storm'. In the present study, the results provided preliminary evidence suggesting that the miR-93-5p-based miRNA therapeutics could be combined with the anti-inflammatory aged garlic extract (AGE) to more effectively inhibit IL-8 gene expression. The human bronchial epithelial IB3-1 cell line was employed as experimental model system. IB3-1 cells were stimulated with the BNT162b2 COVID-19 vaccine and transfected with pre-miR-93-5p in the absence or in the presence of AGE, to verify the inhibitory effects on the BNT162b2-induced expression of the IL-8 gene. The accumulation of IL-8 mRNA was assessed by RT-qPCR; the release of IL-8 protein was determined by Bio-Plex assay. In addition, the possible applications of TLR4/NF-kappa B inhibitory agents (such as miR-93-5p and AGE) for treating human pathologies at a hyperinflammatory state, such as COVID-19, cystic fibrosis and other respiratory diseases, were summarized
Inhibitory effects of SARS-CoV-2 spike protein and BNT162b2 vaccine on erythropoietin-induced globin gene expression in erythroid precursor cells from patients with β-thalassemia
No full textDuring the recent coronavirus disease 2019 (COVID-19) pandemic several patients with β-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with β-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a β-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were β+-IVSI-110/β+-IVSI-110 (one patient), β039/β+-IVSI-110 (3 patients), and β039/ β039 (2 patients). After treatment with S-protein or BNT162b2 for 5 days, lysates were analyzed by high performance liquid chromatography (HPLC), for hemoglobin production, and isolated RNA was assayed by RT-qPCR, for detection of globin gene expression. The main conclusions of the results obtained are that SARS-CoV-2 S-protein and BNT162b2 vaccine (a) inhibit fetal hemoglobin (HbF) production by β-thalassemic ErPCs and (b) inhibit γ-globin mRNA accumulation. In addition, we have performed in silico studies suggesting a high affinity of S-protein to HbF. Remarkably, the binding interaction energy of fetal hemoglobin to S-protein was comparable with that of angiotensin-converting enzyme 2 (ACE2). Our results are consistent with the hypothesis of a relevant impact of SARS-CoV-2 infection and COVID-19 vaccination on the hematopoietic system
Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions
No full textAbstractRapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter
A liquid biopsy model: Circulating miRNA levels in mice bearing colorectal carcinoma tumor xenografts
No full textMicroRNAs (miRNAs) are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This has a very important implication in diagnosis and/or prognosis, including the recent discovery that the pattern of circulating cell-free miRNAs in plasma allows us to perform molecular analyses on these non-invasive liquid biopsies. In order to develop in vivo experimental models for the validation of molecular assays aimed at detecting circulating miRNAs in plasma, we have inoculated nude mice with the HT-29, LS174T and LoVo human colorectal cancer cell lines. Xenotransplanted tumors were allowed to grow up to 0.5 cm3 in size. Blood was drawn at sacrifice, plasma was isolated by low speed centrifugation and was further treated to disrupt exosomes and denaturate miRNA-binding proteins using Qiazol solution. As internal control sequences, equal amount of C. elegans cel-miR-39 was spiked in all samples. Total microRNAs were purified with miRNeasy Serum/Plasma Kit (Qiagen). The levels of miR-141, miR-221 and miR-222 were assessed by Real-Time quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR). Mice xenotransplanted with HT29 cells displayed 2.3, 1.7 and 4.2 fold-increases in circulating miR-141, miR-221 and miR-222, respectively, compared to control mice. Mice transplanted with LoVo cells displayed increases of lesser magnitude (1.2, 1.7 and 2.2 for miR-141, miR-221 and miR-222), whereas mice transplanted with LS174T displayed a slight increase only for miR-222 (1.3 fold-increase). Circulating miRNAs levels were also correlated with their levels in cultured cells and in exosomes from cultured cells supernatants. Although the number of miRNAs taken into account is limited, these data clearly show, in a strictly controlled setting, that different colorectal carcinoma tumors give rise to very different liquid biopsy miRNA profiles. Therefore, wider panels of xenografts may be useful to recapitulate the clinical variety of colorectal tumors in human patients, estimate the minimal numbers of miRNAs in a diagnostic signature, and validate the overall applicative impact of oncomiRNAs in the liquid biopsy format
AGENTI TRASFETTANTI PER ACIDI PEPTIDO-NUCLEICI
No full textL’invenzione riguarda derivati calixarenici funzionalizzati in grado di interagire e legarsi non-covalentemente con acidi peptidonucleici (PNA) e di trasportarli all’interno di cellule. Tali composti possono essere utilizzati come vettori non virali per la trasfezione cellulare di PN
MicroRNAs and Long Non-coding RNAs in Genetic Diseases
Full text linkSince the discovery and classification of non-coding RNAs, their roles have gained great attention. In this respect, microRNAs and long non-coding RNAs have been firmly demonstrated to be linked to regulation of gene expression and onset of human diseases, including rare genetic diseases; therefore they are suitable targets for therapeutic intervention. This issue, in the context of rare genetic diseases, is being considered by an increasing number of research groups and is of key interest to the health community. In the case of rare genetic diseases, the possibility of developing personalized therapy in precision medicine has attracted the attention of researchers and clinicians involved in developing “orphan medicinal products” and proposing these to the European Medicines Agency (EMA) and to the Food and Drug Administration (FDA) Office of Orphan Products Development (OOPD) in the United States. The major focuses of these activities are the evaluation and development of products (drugs, biologics, devices, or medical foods) considered to be promising for diagnosis and/or treatment of rare diseases or conditions, including rare genetic diseases. In an increasing number of rare genetic diseases, analysis of microRNAs and long non-coding RNAs has been proven a promising strategy. These diseases include, but are not limited to, Duchenne muscular dystrophy, cystic fibrosis, Rett syndrome, and β-thalassemia. In conclusion, a large number of approaches based on targeting microRNAs and long non-coding RNAs are expected in the field of molecular diagnosis and therapy, with a facilitated technological transfer in the case of rare genetic diseases, in virtue of the existing regulation concerning these diseases
AGENTI TRASFETTANTI PER microRNA E antagomiRNA
No full textL’invenzione riguarda derivati calixarenici funzionalizzati in grado di interagire e legarsi non-covalentemente con microRNA e antagomiRNA, e di trasportarli all’interno di cellule. Tali composti possono essere utilizzati come vettori non virali per la trasfezione cellulare di microRNA e antagomiRN
Epigenetics and doping in sports—The role of microRNAs
No full textEpigenetic markers have been extensively employed to identify several physiological and/or pathological conditions, as they are associated with a long list of biological processes, including DNA methylation, histone modifications, genomic imprinting, and regulation of protein-coding mRNAs mediated by microRNAs (miRNAs). miRNAs are small noncoding RNAs (15–25 nucleotides in length) able to bind the 3′UTR and, less frequently, 5′UTR and coding sequences (CDS) of target mRNAs leading to mRNA-translation repression and/or mRNA degradation, depending on binding affinity. Recently, miRNAs have been extensively investigated as epigenetic biomarkers for the identification of physiological alterations, such as doping-related procedures, thanks to their easily detection and stability in a large variety of body fluids including bloods and derivatives (plasma and serum). As for several physiological alterations, it is expected that the miRNA expression profile may be altered following the exposure to agents employed in doping procedures. In agreement with this hypothesis circulating miRNAs were found to be associated with administration of human testosterone and growth-hormone and have been proposed as long-term biomarkers for the detection of the erythropoiesis-stimulating agent CERA. Hypoxia-regulated miRNAs might be used to identify the HIF Stabilizer Molidustat. In addition, despite the occurrence of possible confounding factors (changes in the miRNA expression profile during blood storage, aging, exercise, environment), miRNAs have been proposed as potential markers of autologous blood transfusion in sports
Efficient cell penetration and delivery of peptide nucleic acids by an argininocalix[4]arene
Full text linkAbstract The application of Peptide Nucleic Acids (PNAs), mimics of DNA lacking the sugar-phosphate backbone, for antisense/anti-gene therapy and gene editing is limited by their low uptake by cells. Currently, no simple and efficient delivery systems and methods are available to solve this open issue. One of the most promising approach is the modification of the PNA structure through the covalent linkage of poliarginine tails, but this means that every PNA intended to be internalized must be modified. Herein we report the results relative to the delivery ability of a macrocyclic multivalent tetraargininocalix[4]arene (1) used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity and ease preparation of the transfection formulation, simply attained by mixing PNA and calixarene, candidate this vector as universal delivery system for this class of nucleic acid analogues
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