Di Cecilia, Serena
A Novel Long Non-Coding RNA Transcript Regulates The Fate of Colon Cancer Initiation.
[Tesi di dottorato]
Visibile a [TBR] Amministratori dell’archivio fino a 31 Dicembre 2016.
Cancer Initiating cells are a small population of cancer cells capable of tumor initiation and growth. Characteristic of these cells are the expression of “stem-like” markers and the common alteration of the Wnt/β-catenin and Notch pathways. In my thesis, I characterized Colon Cancer Initiating Cells (CCICs) by FACS analysis from HT-29 colon cancer cell line. I then performed in vitro colonospheres-forming assay and RNA-Seq, to interrogate the genome-wide signature involving CCICs. My results show that members of Wnt/β-catenin pathway are elevated in CCICs and colonospheres, when compared to a more differentiated population. Moreover, genes directing the differentiation were silenced in CCICs and colonospheres. These results demonstrate the self-renewal and proliferation capacity of the isolated CCICs population, and that this subpopulation resembles a strong Wnt-signaling pathway signature. Additionally, several lncRNAs are dys-regulated in CCICs, and my findings identify LUST/RBM5-AS1 as a lncRNA transcript strongly elevated during colonospheres formation. The expression of LUST and stem-like markers CD24 and CD44, and expression of Wnt-signaling corresponds with cells that can survive and grow in serum-free media. Loss of LUST impairs Wnt-signaling at mRNA and protein levels, while LUST overexpression provides enhanced and synergistic signaling mediated through Wnt and β-catenin at the mRNA and protein levels. Nuclear/Cytoplasmic RNA fractionation and RNA-FISH show that LUST essentially is a nuclear transcript. RNA immuno-precipitation and UV cross-linking immuno-precipitation assays show that LUST RNA binds to β-catenin. Finally, LUST overexpression enhances colonospheres formation more rapidly. Collectively, my findings reveal that the lncRNA LUST regulates Wnt pathway in CCICs through a coordinated physical interaction with β-catenin, acting through transcriptional regulation to promote colon cancer initiating cells maintenance.
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