Investigation into the mechanistic effects of the lncRNA Growth Arrest Specific 5 in the regulation of cell survival

Maroszek, Maria Regina

Abstract

Current research indicates that long noncoding RNAs orchestrate various regulatory processes in a physiological context and thereby, their dysregulation may contribute to major human diseases, including cancer (Grammatikakis et al.,2014).

Long noncoding RNA (LncRNA) is a large class of RNA molecules with size larger than 200 nucleotides, exhibit cellular functions although having no protein-coding capability, are important regulators of the epigenetic status of the human genome (Uchida and Bolli, 2018). Emerging evidence has shown that lncRNAs are important regulators in gene expression networks by controlling nuclear architecture at epigenetic, transcriptional level in the nucleus by modulating mRNA stability, and posttranscriptional modifications in the cytoplasm (Statello et al.,2020). Moreover, recent evidence indicates that their mode of action is not only limited to the molecular levels but also these non-protein coding molecules modulate several cellular functions, for instance: proliferation, apoptosis, invasion, and metastasis (Chowdhary et al.,2021 & Yao et.al.,2019). Besides their participation to normal physiology, lncRNA expression and function have been already associated to many diseases, including cancer (Morlando and Fatica,2018). Consequently, detailed research in this matter may offer new insights into pathogenetic mechanisms and thereby offer a novel approach to diagnosis and therapy. Of particular interest in this regard is Growth Arrest Specific Transcript (GAS5) lncRNA, which has been shown to function as a tumour suppressor and confirmed to be downregulated in multiple cancers, with expression levels related to both clinical characteristics and patient prognosis (Yu and Hann,2019). Previous GAS5-related studies identified three separate structural modules that act independently in leukemic T cells (Frank et al.,2020). Consistently, functional assays in this study such as MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2- (4sulfophenyl)-2H-tetrazolium) and the Muse® Annexin V and Dead Cell assay enabled to determine viable and apoptotic cells number as well as the percentage of cells in each stage of the cell cycle. These assays allow to investigate the role of GAS5 structural modules in the regulation of leukemic cell survival.

The outcome from this investigation indeed confirmed that to fully exhibit its antitumor potential, GAS5 requires all the structural effector molecules to be present to form a binding platform complex, this is in turn may explain why the full-length construct indicated the highest antiproliferation and pro-apoptotic potential as well as to induce cell cycle arrest. The other constructs seemed to influence the cell viability and apoptosis but were considerably less significant than those seen in the full-length construct. In this study construct with the nucleotide sequence 1-167 which corresponds to 5′ terminal, reduced the number and percentage of the viable cell, indicated a significant effect on the percentage of apoptotic cells as well as induce a cell cycle arrest. Consistently, constructs with the nucleotide sequences 185-252 and 475-531 which correspond to the core modules seemed to influence the number and percentage of the viable cell as well as apoptosis. The study confirmed that the 5' terminal module with a low secondary structure affects basal survival and slows the cell cycle, whereas the highly structured core module also affects basal survival at some extent but mainly mediates the effects of mammalian target of rapamycin (mTOR) inhibition on cell growth.

The outcomes from this investigation revealed that different parts of GAS5 lncRNA exert different functions in T- cell leukemic cells. Therefore, this complex cellular mechanism is likely to form the basis of GAS5 tumour suppressor action. These results highlight the predominant role of GAS5 in regulating cell survival and reveal how a single lncRNA transcript utilizes a modular structure-function relation to respond to a variety of cellular stresses under various cellular conditions.

Citation

Maroszek, M. R. Investigation into the mechanistic effects of the lncRNA Growth Arrest Specific 5 in the regulation of cell survival. (Thesis). Keele University. https://keele-repository.worktribe.com/output/884975

• Thesis Type: Thesis
• Deposit Date: Aug 16, 2024
• Publicly Available Date: Aug 16, 2024
• Public URL: https://keele-repository.worktribe.com/output/884975
• Award Date: 2024-08