The role of long non-coding RNA LINC00673 in lung tumorigenesis

Abstract

Eukaryotic cellular processes are governed by extensive regulatory networks involving multiple cellular species. Non-coding RNAs (ncRNAs) are a class of RNA molecules varying in size, biogenesis and mechanism of action that have established themselves as key regulators of cellular behaviour. This project explores the roles of long non-coding RNA (lncRNA) in the progression of lung adenocarcinoma (LUAD) – the most common type of lung cancer – to improve current understanding of the disease. Using a combination of computational and wet-lab approaches, we identified LINC00673 as a potential regulator of LUAD tumorigenesis. RNA sequencing data from three different studies showed LINC00673 is among the top upregulated lncRNAs in patient tumours. Further bioinformatic analysis suggested expression of LINC00673 in tumour samples strongly correlates with the mRNA levels of SOX9, a major developmental regulator and oncogene in the lung context. Recently, evidence has emerged that developmental pathways are re-activated during LUAD progression. We hypothesized that LINC00673 might be involved in those phenomena through regulatory links with SOX9. Indeed, LINC00673 and SOX9 are found in close genomic proximity, and publicly available Hi-C data suggests contacts between the two loci. The genomic region of LINC00673 and SOX9 is syntenic in vertebrates, further supporting the idea of functional links between the two. LINC00673 displays nuclear enrichment in lung cancer cell lines with an apparent accumulation at its locus of transcription hinting at a potential function in cis. Interestingly, locked nucleic acid antisense oligonucleotide (LNA ASO) knockdown of LINC00673 enhances cell growth contrary to our hypothesis that the lncRNA functions as an oncogene. Knock-down of LINC00673 by CRISPRi and LNA ASOs suggest that downregulation of the lncRNA at the transcriptional level results in upregulation of SOX9 mRNA and protein. The mRNA levels of other nearby genes are also increased suggesting LINC00673 regulates transcription of the whole genomic region. Nevertheless, the presence of the mature lncRNA is not required for this transcriptional control since overexpression of LINC00673 does not alter the levels of the genes tested. Together these results suggest LINC00673 is a cis-acting lncRNA regulating transcription in its genomic region. The mechanism of action might involve enhancers or other regulatory elements whose accessibility is altered depending on the transcriptional state of the lncRNA, but further work will be required to investigate this hypothesis. With this project we aim to better understand the regulatory roles of lncRNAs in health and disease. With the recent advances in the field of RNA therapeutics, lncRNAs are now druggable targets. Improved understanding of lncRNA biology and function can lead to the development of novel therapeutic strategies to treat various cancers, the identification of biomarkers and better patient stratification for personalised medicine.