The biological importance of the non-coding transcriptome has become indisputable over the years. The relative proportion of the protein-encoding genome compared to the regulatory non-protein encoding genome varies significantly among species, indicating a relationship between the complexity of higher eukaryotes and non-coding RNAs (ncRNAs). RNAs, especially ncRNAs, are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Interestingly, complex diseases like cancer frequently involve quantitative changes to the non-coding transcriptome, suggesting a potential link between ncRNA dysfunction and pathological phenotypes. Prostate cancer (PCa), a complex and multifaceted disease, involves the dysregulation of various molecular processes, including the intricate realm of ncRNAs. Indeed, altered expression patterns of ncRNAs have been observed in PCa, contributing to the disease's initiation, progression, and treatment response. Some ncRNAs act as key regulators, influencing critical cellular functions like proliferation, apoptosis, and invasion. Moreover, specific ncRNAs have emerged as potential diagnostic and prognostic markers, offering valuable insights into the aggressiveness of the tumor and guiding personalized treatment strategies. PCa constitutes a substantial global health concern, evidenced by the yearly diagnosis of approximately 1.3 million novel cases on a global scale; understanding the complex network interplay between PCa and ncRNAs holds promise for uncovering novel therapeutic targets and advancing precision medicine approaches in the management of this prevalent malignancy. Commencing from these concepts, we have developed the work outlined in this thesis, focusing on two main objectives: 1) Study of potential RNA biomarkers for PCa. 2) Investigation of intracellular networks involving ncRNAs. In relation to the first objective, our study has provided a significant contribution to the European project diaRNAgnosis H2020, aiming at the development of a PCR-free and non-invasive diagnosis of PCa through the detection of RNA biomarkers in biological fluids. In this context, in line with the work packages in which we are involved, we optimized the protocols for urine sample manipulation and RNA extraction. Additionally, we analysed, selected, and proposed 3 lncRNAs (MALAT1, PCA3, PCAT18), one mRNA (KLK3), and 8 microRNAs (miR-27b-3p, miR-30a-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-107, miR-125b-5p, miR-574-3p) as potential biomarkers for PCa, especially capable of discriminating patients with cancer from those with Benign Prostatic Hyperplasia (BPH). During secondment periods at the biotech company DestiNA Genomica SL (Granada, Spain), the selected transcripts were then utilized for the design of probes to be employed in the development of the diagnostic device. Further tests and validations are underway for the device's development. Concerning the study of intracellular networks, we employed a combined approach of computational and experimental biology to identify deregulated lncRNAs in PCa. Expression analysis on PCa biopsies of lncRNAs previously selected through computational analysis enabled the identification of a statistically significantly overexpressed lncRNA – ELFN1-AS1 – in tumor tissues compared to adjacent healthy tissue. Subcellular localization studies, defining it as preferentially cytoplasmic led us to hypothesize its function as a "miRNA sponge" and its involvement in competing endogenous RNA (ceRNA) networks, such as the in silico-identified ELFN1-AS1:miR-139-3p-CRISP3 network. We did not exclude its involvement in regulatory processes within the nucleus by investigating potential RNA:RBP interactions. In conclusion, the obtained results encourage further exploration of the intricate networks in which ELFN1-AS1 is involved through interactions with other biomolecules in the complex system of PCa.
L'importanza biologica del trascrittoma non codificante è diventata indiscutibile nel corso degli anni. La proporzione relativa del genoma che codifica per le proteine rispetto al genoma non codificante regolatorio varia significativamente tra le specie, indicando una relazione tra la complessità degli eucarioti superiori e gli RNA non codificanti (ncRNA) che hanno una funzione regolatoria. Gli RNA, specialmente gli ncRNA, sono attori cruciali nella regolazione dei meccanismi cellulari grazie alla loro capacità di interagire e regolare altre molecole. È interessante notare che malattie complesse come il cancro coinvolgono frequentemente cambiamenti quantitativi nel trascrittoma non codificante, suggerendo un possibile legame tra deregolazione degli ncRNA e fenotipi patologici. Il cancro alla prostata (PCa), una malattia complessa e sfaccettata, coinvolge la deregolazione di vari processi molecolari, inclusa l'intricata sfera degli ncRNA. Infatti, sono stati osservati pattern di espressione alterati degli ncRNA nel PCa, contribuendo all'inizio, alla progressione e alla risposta al trattamento della malattia. Alcuni ncRNA agiscono come regolatori chiave, influenzando funzioni cellulari critiche come la proliferazione, l’apoptosi e l’invasione. Inoltre, è emerso il ruolo di specifici ncRNA come potenziali marcatori diagnostici e prognostici, offrendo preziosi insights sulla aggressività del tumore e guidando strategie di trattamento personalizzate. Il PCa costituisce una notevole preoccupazione per la salute globale, come dimostrato dalla diagnosi annuale di circa 1,3 milioni di nuovi casi su scala mondiale; comprendere l'interazione delle complesse network tra PCa e ncRNA offre la possibilità di scoprire nuovi bersagli terapeutici e sviluppare approcci basati sulla medicina di precisione nella gestione di questo tumore molto diffuso. Partendo da questi concetti, abbiamo sviluppato il lavoro proposto in questa tesi, concentrandoci su due obiettivi principali: 1)Studio di potenziali RNA biomarcatori del PCa. 2)Studio delle network intracellulari in cui sono coinvolti gli ncRNA. Riguardo al primo obiettivo, il nostro studio ha fornito un contributo significativo al progetto europeo diaRNAgnosis H2020, mirato allo sviluppo di una diagnosi del PCa non invasiva e senza l’utilizzo della PCR, attraverso il rilevamento di RNA biomarcatori nei fluidi biologici. In questo contesto, in linea con i work flow in cui siamo coinvolti, abbiamo ottimizzato un protocollo per la manipolazione del campione di urina e l'estrazione di RNA. Inoltre, abbiamo analizzato, selezionato validato e proposto 3 lncRNA (MALAT1, PCA3, PCAT18), un mRNA (KLK3) e 8 microRNA (miR-27b-3p, miR-30a-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-107, miR-125b-5p, miR-574-3p) come potenziali biomarcatori per il PCa, in grado anche di discriminare i pazienti affetti da cancro da quelli affetti da iperplasia prostatica benigna (BPH). Durante i periodi di secondment presso l'azienda biotecnologica DestiNA Genomica SL (Granada, Spagna), i trascritti selezionati sono stati utilizzati per la progettazione di sonde da impiegare nello sviluppo del dispositivo diagnostico. Ulteriori test e validazioni sono in corso per lo sviluppo del dispositivo. Per quanto riguarda lo studio delle network intracellulari, abbiamo utilizzato un approccio combinato di biologia computazionale e sperimentale per identificare i lncRNA deregolati nel PCa. L'analisi dei livelli di espressione in biopsie di PCa dei lncRNA precedentemente selezionati tramite analisi computazionale ha permesso l'identificazione di un lncRNA sovraespresso in modo statisticamente significativo - ELFN1-AS1 - nei tessuti tumorali rispetto al tessuto sano adiacente. Studi sulla localizzazione subcellulare, lo hanno localizzato come preferenzialmente citoplasmatico, ci hanno portato a ipotizzare la sua funzione di "miRNA sponge" e il suo coinvolgimento all’interno di ceRNA, come la network ELFN1-AS1:miR-139-3p-CRISP3 identificata in silico. Non è stato escluso il suo coinvolgimento in processi regolatori nucleari studiando potenziali interazioni RNA:RBP. In conclusione, i risultati ottenuti incoraggiano ulteriori approfondimenti sullo studio delle intricate network in cui ELFN1-AS1 è coinvolto attraverso interazioni con altre biomolecole nel sistema complesso del PCa.
ncRNA come biomarcatori e regolatori molecolari del cancro alla prostata
STELLA, MICHELE
2024
Abstract
The biological importance of the non-coding transcriptome has become indisputable over the years. The relative proportion of the protein-encoding genome compared to the regulatory non-protein encoding genome varies significantly among species, indicating a relationship between the complexity of higher eukaryotes and non-coding RNAs (ncRNAs). RNAs, especially ncRNAs, are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Interestingly, complex diseases like cancer frequently involve quantitative changes to the non-coding transcriptome, suggesting a potential link between ncRNA dysfunction and pathological phenotypes. Prostate cancer (PCa), a complex and multifaceted disease, involves the dysregulation of various molecular processes, including the intricate realm of ncRNAs. Indeed, altered expression patterns of ncRNAs have been observed in PCa, contributing to the disease's initiation, progression, and treatment response. Some ncRNAs act as key regulators, influencing critical cellular functions like proliferation, apoptosis, and invasion. Moreover, specific ncRNAs have emerged as potential diagnostic and prognostic markers, offering valuable insights into the aggressiveness of the tumor and guiding personalized treatment strategies. PCa constitutes a substantial global health concern, evidenced by the yearly diagnosis of approximately 1.3 million novel cases on a global scale; understanding the complex network interplay between PCa and ncRNAs holds promise for uncovering novel therapeutic targets and advancing precision medicine approaches in the management of this prevalent malignancy. Commencing from these concepts, we have developed the work outlined in this thesis, focusing on two main objectives: 1) Study of potential RNA biomarkers for PCa. 2) Investigation of intracellular networks involving ncRNAs. In relation to the first objective, our study has provided a significant contribution to the European project diaRNAgnosis H2020, aiming at the development of a PCR-free and non-invasive diagnosis of PCa through the detection of RNA biomarkers in biological fluids. In this context, in line with the work packages in which we are involved, we optimized the protocols for urine sample manipulation and RNA extraction. Additionally, we analysed, selected, and proposed 3 lncRNAs (MALAT1, PCA3, PCAT18), one mRNA (KLK3), and 8 microRNAs (miR-27b-3p, miR-30a-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-107, miR-125b-5p, miR-574-3p) as potential biomarkers for PCa, especially capable of discriminating patients with cancer from those with Benign Prostatic Hyperplasia (BPH). During secondment periods at the biotech company DestiNA Genomica SL (Granada, Spain), the selected transcripts were then utilized for the design of probes to be employed in the development of the diagnostic device. Further tests and validations are underway for the device's development. Concerning the study of intracellular networks, we employed a combined approach of computational and experimental biology to identify deregulated lncRNAs in PCa. Expression analysis on PCa biopsies of lncRNAs previously selected through computational analysis enabled the identification of a statistically significantly overexpressed lncRNA – ELFN1-AS1 – in tumor tissues compared to adjacent healthy tissue. Subcellular localization studies, defining it as preferentially cytoplasmic led us to hypothesize its function as a "miRNA sponge" and its involvement in competing endogenous RNA (ceRNA) networks, such as the in silico-identified ELFN1-AS1:miR-139-3p-CRISP3 network. We did not exclude its involvement in regulatory processes within the nucleus by investigating potential RNA:RBP interactions. In conclusion, the obtained results encourage further exploration of the intricate networks in which ELFN1-AS1 is involved through interactions with other biomolecules in the complex system of PCa.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/165717
URN:NBN:IT:UNICT-165717