Splicing is key mechanism involved in the post-transcriptional regulation of gene expression in eukaryotic cells. For protein coding genes, the primary transcript (pre-mRNA) is composed of both coding sequences, called exons, and non-coding regions known as introns. The splicing process allows the removal of introns, thus generating a mature transcript (mRNA) that is translated in the cytoplasm. However, for many genes splicing occurs in a regulated form and this process, which is known as alternative splicing (AS), generates different mature mRNAs from the same pre-mRNA. AS comprehends a series of molecular events that are able to sustain the expression of diverse functional protein isoforms. Remarkably, 95% of the human genes are regulated through AS, which is therefore responsible for greatly increasing proteome diversity. Given the importance of AS, it is not surprising that its alterations are involved in several human diseases, including cancer development and progression. In the past our laboratory showed that the tissue-specific AS regulator NOVA2, for a long time considered expressed only in the neural cells of the central nervous system, is also expressed in endothelial cells (ECs) of different organs. We demonstrated that NOVA2 regulates angiogenesis and vascular development at post-transcriptional level. In addition, we identified novel genes regulated through NOVA2-mediated AS in ECs. Recently, we have also demonstrated that NOVA2 is selectively over-expressed in the tumor vasculature of many cancer types, including ovarian, colorectal, liver hepatocellular, and head-neck squamous cell cancers, with negligent expression in the others cell types within the tumor. Notably, in ovarian and colorectal cancer patients, we found that NOVA2 has a prognostic value since its expression levels correlate with shorter overall survival of the cancer patients. The main goal of my thesis was to identify transcription factor(s) regulating NOVA2 expression in ECs. Several preliminary data generated in my laboratory have supported a role for the transcription factor ZEB1 as a putative NOVA2 activator in ECs. In order to demonstrate that ZEB1 is required for NOVA2 expression, I performed its siRNA-mediated knockdown in human Umbelical Vein Endothelial Cells (HUVECs). By performing RT-qPCR and Western blot analyses, I found that ZEB1 depletion significantly reduced NOVA2 expression at both RNA and protein levels. Following ZEB1 downregulation, I also observed changes in the AS profile of selected NOVA2 target genes in HUVECs. Interestingly, ZEB1 depletion impaired in vitro angiogenesis, as evaluated by a tube formation assay, like NOVA2. In order to investigate if factors secreted from tumor cells are able to activate the ZEB1/NOVA2 circuit in ECs, I treated HUVECs with conditional medium (CM) from ovarian cancer cells. Importantly, after CM treatment, I observed a significant increase in ZEB1 and NOVA2 expression levels as well as changes in NOVA2 splicing, which were coherent with the upregulation of this AS factor in tumor vasculature. Collectively, my data reveal the existence of a novel pathway that could be involved in the phenotypic and functional aberrancies of tumor blood vessels.
Lo splicing è un meccanismo essenziale coinvolto nella regolazione post-trascrizionale dell’espressione genica negli eucarioti. Per i geni codificanti per proteine, il trascritto primario (pre-mRNA) è composto sia da sequenze codificanti, chiamate esoni, sia da sequenze non codificanti note come introni. Il processo di splicing consiste nella rimozione degli introni generando il trascritto finale maturo (mRNA) che viene successivamente tradotto nel citoplasma. Molti geni sono però soggetti ad una forma particolare di splicing, nota come splicing alternativo (AS), in grado di generare diversi mRNA maturi a partire dallo stesso pre-mRNA. AS comprende una serie di eventi diversi tra loro in grado di supportare l’espressione di diverse isoforme proteiche. E’ importante sottolineare che il 95% dei geni umani sono regolati tramite AS, che risulta di conseguenza fondamentale per generare la diversità del proteoma delle nostre cellule. Data la sua rilevanza, non è sorprendente che alterazioni nella reazione di AS siano coinvolte in molte malattie umane ed in particolare nello sviluppo e la progressione tumorale. In passato il nostro laboratorio ha dimostrato che il regolatore di AS tessuto-specifico NOVA2, a lungo considerato espresso esclusivamente nei neuroni del sistema nervoso centrale, è anche espresso nelle cellule endoteliali che compongono i vasi sanguigni in diversi organi. Abbiamo dimostrato che NOVA2 regola l’angiogenesi e lo sviluppo del sistema vascolare a livello post-trascrizionale. Inoltre, abbiamo identificato nuovi geni il cui AS è regolato da NOVA2 nelle cellule endoteliali. Recentemente abbiamo anche scoperto che NOVA2 è selettivamente over-espressa nella vascolatura tumorale di diversi tipi di cancro, incluso il tumore ovarico, al colon-retto, epatocellulare, carcinomi a cellule squamose della testa e del collo. Al contrario, la sua espressione è pressoché nulla negli altri tipi cellulari presenti all’interno del tumore. Notevolmente, nei pazienti affetti da tumore ovarico e al colon-retto abbiamo osservato che NOVA2 ha un valore prognostico, dal momento che la sua espressione è correlata con una ridotta sopravvivenza dei pazienti oncologici. Il principale obiettivo della mia tesi è stato quello di identificare fattori di trascrizioni coinvolti nella regolazione dell’espressione di NOVA2 nelle cellule endoteliali. Diversi dati preliminari prodotti nel mio laboratorio hanno indicato il fattore di trascrizione ZEB1 come possibile attivatore di NOVA2 nelle cellule endoteliali. Per dimostrare che ZEB1 è necessario per l’espressione di NOVA2, ho indotto una sua riduzione mediante siRNA nelle Umbelical Vein Endothelial Cells (HUVECs). Dopo aver eseguito analisi mediante RT-qPCR e Western blot, ho scoperto che la downregolazione di ZEB1 induceva una significativa riduzione dell’espressione di NOVA2 sia a livello di trascritto che di proteina. A seguito della downregolazione di ZEB1, ho anche osservato cambiamenti nei profili di AS di selezionati targets di splicing di NOVA2 nelle cellule endoteliali. La rimozione di ZEB1, come quella di NOVA2, è stata inoltre in grado di influire negativamente sull’angiogenesi in vitro, come dimostrato dagli esperimenti di formazione dei tubi. Per studiare se eventuali fattori secreti dalle cellule tumorali fossero in grado di attivare il circuito ZEB1/NOVA2 nelle cellule endoteliali, ho trattato le HUVECs con terreno di coltura condizionato (CM) da cellule di tumore ovarico. Dopo il trattamento con CM, ho osservato nelle HUVECs un aumento significativo dell’espressione di ZEB1 e NOVA2, così come un cambiamento negli eventi di splicing regolati da NOVA2, con un andamento che era coerente con a quello osservato nei vasi tumorali con incrementata espressione di NOVA2. Collettivamente, i miei dati rivelano l’esistenza di una nuova via di segnalazione che potrebbe essere coinvolta nel sostenere lo sviluppo delle anomalie dei vasi tumorali.
Study of NOVA2/ZEB1 circuit in endothelial cells
RECUSANI, MATILDE
2021/2022
Abstract
Splicing is key mechanism involved in the post-transcriptional regulation of gene expression in eukaryotic cells. For protein coding genes, the primary transcript (pre-mRNA) is composed of both coding sequences, called exons, and non-coding regions known as introns. The splicing process allows the removal of introns, thus generating a mature transcript (mRNA) that is translated in the cytoplasm. However, for many genes splicing occurs in a regulated form and this process, which is known as alternative splicing (AS), generates different mature mRNAs from the same pre-mRNA. AS comprehends a series of molecular events that are able to sustain the expression of diverse functional protein isoforms. Remarkably, 95% of the human genes are regulated through AS, which is therefore responsible for greatly increasing proteome diversity. Given the importance of AS, it is not surprising that its alterations are involved in several human diseases, including cancer development and progression. In the past our laboratory showed that the tissue-specific AS regulator NOVA2, for a long time considered expressed only in the neural cells of the central nervous system, is also expressed in endothelial cells (ECs) of different organs. We demonstrated that NOVA2 regulates angiogenesis and vascular development at post-transcriptional level. In addition, we identified novel genes regulated through NOVA2-mediated AS in ECs. Recently, we have also demonstrated that NOVA2 is selectively over-expressed in the tumor vasculature of many cancer types, including ovarian, colorectal, liver hepatocellular, and head-neck squamous cell cancers, with negligent expression in the others cell types within the tumor. Notably, in ovarian and colorectal cancer patients, we found that NOVA2 has a prognostic value since its expression levels correlate with shorter overall survival of the cancer patients. The main goal of my thesis was to identify transcription factor(s) regulating NOVA2 expression in ECs. Several preliminary data generated in my laboratory have supported a role for the transcription factor ZEB1 as a putative NOVA2 activator in ECs. In order to demonstrate that ZEB1 is required for NOVA2 expression, I performed its siRNA-mediated knockdown in human Umbelical Vein Endothelial Cells (HUVECs). By performing RT-qPCR and Western blot analyses, I found that ZEB1 depletion significantly reduced NOVA2 expression at both RNA and protein levels. Following ZEB1 downregulation, I also observed changes in the AS profile of selected NOVA2 target genes in HUVECs. Interestingly, ZEB1 depletion impaired in vitro angiogenesis, as evaluated by a tube formation assay, like NOVA2. In order to investigate if factors secreted from tumor cells are able to activate the ZEB1/NOVA2 circuit in ECs, I treated HUVECs with conditional medium (CM) from ovarian cancer cells. Importantly, after CM treatment, I observed a significant increase in ZEB1 and NOVA2 expression levels as well as changes in NOVA2 splicing, which were coherent with the upregulation of this AS factor in tumor vasculature. Collectively, my data reveal the existence of a novel pathway that could be involved in the phenotypic and functional aberrancies of tumor blood vessels.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15510