Novel RNA-based biomarkers for ovarian cancer – uncovering how LINC01132 is regulated by the tumour suppressor protein, p53

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Hartigan, Shaun
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University College Cork
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Ovarian cancer is one of the deadliest female cancers worldwide. Most cases are detected in advanced stages, as it is difficult to diagnose ovarian cancer early due to nonspecific symptoms. Presently, there are no sensitive biomarkers to identify early-stage disease. Over 60% of ovarian cancer cases have a mutation in p53, a tumour suppressor transcription factor which counteracts cell stress and oncogenic signals. Most mutations occur within p53’s DNA-binding domain, a vital region which facilitates its anchorage to target gene promoters. Previous data found numerous long-non-coding RNAs (lncRNAs) are differentially expressed in ovarian cancer cells with mutant TP53, compared to those with the wildtype gene. Here, we show that three p53 mutants (R175H, I195T and R248Q) commonly found in ovarian cancer patients, were unable to activate firefly luciferase expression from a synthetic p53-responsive promoter, reflecting the impact of p53 core domain mutations on its ability to bind target promoters and transactivate gene expression. In silico genome-wide ChIP-seq analysis of differentially expressed lncRNAs identified three (MEG3, LINC01132, and LINC2960) containing regions within -1 kb of their transcription start sites, showing interaction with p53. Another four (EMX20S, PRICKLE2-DT, LINC00887 and LINC02610 contained p53-interacting regions within -5 kb. Division of the region up to -6,995 bp of the LINC01132 transcription start site into a distal, middle, and proximal segment, and subsequent cloning upstream of firefly luciferase, allowed us to assess p53 activity at the LINC01132 promoter. Western blot analysis could not detect luciferase expression from either segment under wildtype p53 overexpression, despite the proximal segment containing six p53-interacting sites. To determine the true response of wildtype and mutant p53 binding to the LINC01132 promoter, future quantitative reverse-transcriptase PCR and luciferase assays should be conducted, given their higher sensitivity compared to Western blot analysis. To improve patient prognosis in ovarian cancer, there is vital necessity to discover specific biomarkers, which can diagnose and monitor disease progression. LncRNAs can be detected in blood, so linking expression of differentially expressed lncRNAs to the mutational status of p53 in ovarian cancer, and studying how these change with the therapies, is novel, previously unexplored, and may aid in biomarker discovery.
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Ovarian cancer , Long non-coding RNAs , p53 , Promoters , Regulation
Hartigan, S. W. 2023. Novel RNA-based biomarkers for ovarian cancer – uncovering how LINC01132 is regulated by the tumour suppressor protein, p53. MRes Thesis, University College Cork.
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