Analysis of the effects of non-coding variants in carcinogenesis

Postgraduate Thesis uoadl:2957597 162 Read counter

Unit:
Specialty Molecular Biomedicine Mechanisms of Disease, Molecular and Cellular Therapies, and Bioinnovation
Library of the School of Health Sciences
Deposit date:
2021-07-21
Year:
2021
Author:
Vasilogianni Maria-Evangelia
Supervisors info:
Παντελής Χατζής, Ερευνητής B’, Ίδρυμα βασικής βιοϊατρικής έρευνας, Ε.ΚΕ.Β.Ε. «ΑΛΕΞΑΝΔΡΟΣ ΦΛΕΜΙΓΚ»
Αντιγόνη Δήμα, Ερευνήτρια Γ’, Ίδρυμα βασικής βιοϊατρικής έρευνας, Ε.ΚΕ.Β.Ε. «ΑΛΕΞΑΝΔΡΟΣ ΦΛΕΜΙΓΚ»
Αριστείδης Ηλιόπουλος, Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Original Title:
Analysis of the effects of non-coding variants in carcinogenesis
Languages:
English
Translated title:
Analysis of the effects of non-coding variants in carcinogenesis
Summary:
The intestinal epithelium is the most actively renewed tissue of the human body. The outstanding capacity for renewal of this tissue is attributed to intestinal epithelial stem cells residing in the bottom of the intestinal crypts. These cells are controlled by the signals they receive from their surrounding microenvironment. One of the major contributors to intestinal epithelial stem cell maintenance, differentiation, and proliferation is the Wnt/β-cat pathway. Several studies have shown that mutations in components of the Wnt/β-cat pathway leading to its over-activation, are linked to aberrant cell proliferation in the intestinal epithelium and colorectal cancer.
Even though the Wnt/β-cat pathway has been studied extensively, there are aspects of its regulation that are yet to be elucidated. It has been shown that TCF4 and β-catenin, the transcriptional effectors of the canonical Wnt pathway, are recruited to non-coding areas of the genome, which include various lncRNAs. This heterogeneous class of molecules, which are defined as having a length >200 nucleotides and do not generally code for proteins, can function in a variety of ways. One methodology used to understand the role of a lncRNA in cellular homeostasis and disease involves employing loss of function approaches. Therefore, in this study, we take advantage of the CRISPR/Cas9 system to knock-out WiNTRLINC2 in Ls174 cells. WiNTRLINC2 is a lncRNA whose expression is regulated by the Wnt/β-cat pathway, and which is overexpressed in colorectal cancer patient samples. More specifically, we generated stable WiNTRLINC2 KO cell lines that lack either the promoter or the first exon of WiNTRLINC2, attempting to understand the contribution of the act of transcription of WiNTRLINC2 and the WiNTRLINC2 transcript itself to cellular homeostasis and carcinogenesis. Subsequent expression analyses of the generated KO cell lines indicated a list of 418 common deregulated genes between the two KO strategies. These genes are implicated in various biological processes and primarily the cell cycle, suggesting that WiNTRLINC2 may contribute to cancer manifestation through the regulation of the cell cycle.
In addition, we sought to explore and identify WiNTRLINC2 regulatory regions, as well as regulatory regions of other lncRNAs and genes that are important in carcinogenesis by making use of a bacterial artificial chromosome-self-transcribing active regulatory region (BAC-STARR)-seq approach. Within this framework, we transfect a multitude of cancer cell lines, representing different cancer backgrounds, with a STARR library containing genomic areas in order to identify prospective regulatory elements that regulate the expression of those genes. The results produced are expected to give answers to many questions regarding the differential regulation of WiNTRLINC2 and other lncRNAs and cancer-associated genes of interest in the context of various cancer types, as well as point out genetic variants in the non-coding genome with regulatory and biomarker relevance.
Main subject category:
Health Sciences
Keywords:
LncRNAs, Long non-coding RNAs, Wnt/b-cat signaling pathway, Wnt pathway, Carcinogenesis, KO, Loss of function, WiNTRLINC2, Enhancers, STARR-seq
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of references:
189
Number of pages:
67
File:
File access is restricted only to the intranet of UoA.

Maria Evangelia Vasilogianni_MScThesis.pdf
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File access is restricted only to the intranet of UoA.