Identification and characterisation of non-coding RNAs involved in ductal carcinoma in situ

Ductal carcinoma in situ (DCIS) is an early form of breast cancer, the most common cancer in women worldwide. DCIS can develop into potentially fatal invasive disease, but studies show that less than half of all cases progress if untreated. Currently, most patients receive surgery, as there is no reliable way to distinguish which cases will become invasive. The goal of this work is to contribute to developing a molecular-based method that can differentiate indolent and potentially invasive DCIS. To do this, I identified non-coding ribonucleic acids (ncRNAs) that are involved in DCIS and aimed to discover their molecular functions. Through target-capture RNA sequencing, I identified 181 ncRNAs overexpressed in high-grade DCIS cell lines (ETCC-006 and ETCC-010), compared to normal-like breast cells. Next, the specificity of the ncRNAs was validated across a panel of cancer cell lines. From that, two candidates were selected for in-depth analysis: vtRNA2-1 and MEG3. Their expression was also measured in a small cohort of DCIS patients (n=12) and the overexpression observed in cell lines was confirmed. Vault RNA 2-1 (vtRNA2-1) is an RNA that has been implicated in some cancers; however, its mechanism of action is not clear. Through bisulfite sequencing I found it was hypomethylated in DCIS cell lines, driving its high expression levels. Antisense oligonucleotide knockdown resulted in reduced migration and overexpression increased proliferation in DCIS cells, suggesting it may act as an oncogene. Using RNA-affinity chromatography and UV-crosslinking, followed by mass spectrometry, protein interactors of vtRNA2-1 were identified. These showed enrichment in cytoskeletal processes. Furthermore, I examined genes with altered expression following knockdown of vtRNA2-1, and many of these were associated with various cancer processes. Taken together, my findings suggest an oncogenic role for vtRNA2-1 in the context of DCIS. Maternally expressed gene 3 (MEG3) is a tumour-suppressive long ncRNA that is usually downregulated in various cancers; however, I found that this RNA was highly expressed in DCIS cell lines and patient samples. Like vtRNA2-1, MEG3 was epigenetically regulated in DCIS cell lines. Previous observations that its knockdown increases proliferation and suppresses overexpression were confirmed. However, MEG3 appeared to promote migration, in contrast to what has been shown in breast cancer previously. I confirmed MEG3 to be primarily nuclear and examined genes altered after its knockdown. From gene set enrichment analysis (GSEA), pathways related to the cell cycle and neuronal maturation were prominent. Overall, MEG3 appears to be tumour suppressive in DCIS and could be a marker of indolent disease. By discovering these ncRNAs found in abundance in high-grade DCIS, but not in normal-like or invasive breast cancer cell lines and showing that they have a role in the cancer characteristics of DCIS, I believe these RNAs could eventually be used as biomarkers to stratify patients.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2025

Author dc:creator

• Derlipanska, Magdalina

Advisor dc:contributor.advisor

• Dean, Kellie

• DCIS
• Ductal carcinoma in situ
• ncRNA
• Non-coding RNA
• Breast cancer
• lncRNA

Statement dc:rights

• © 2025, Magdalina Derlipanska.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/18450

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/18450