Subcellular Localization of uc.8+ as a Prognostic Biomarker in Bladder Cancer Tissue

SIMPLE SUMMARY: DNA regions having high sequence similarity among human, rat and mouse genomes are defined as Ultraconserved Regions. Non-coding RNA transcripts originating by these regions may play relevant roles in the onset and progression of multiple cancer types. We recently found that ultra-conserved-transcript-8+ (uc.8+) levels correlate with the grading and staging of bladder cancer. The aim of this study is to systematically evaluate the expression of ultra-conserved-transcript-8+ (uc.8+) in biopsies and assess its intracellular localization. Furthermore, we aimed to correlate uc.8+ levels with clinical parameters and patient survival. Our analysis indicates that uc.8+ can localize both in the cytoplasm and nucleus of bladder cells at early stages of tumorigenesis, while in tumors at advanced stages, uc.8+ has a prevalent cytoplasmic localization. These data provide relevant information about uc.8+ localization as a hallmark of tumor stage. Finally, using advanced computer-based techniques, we predicted the binding of uc.8+ to RNA-binding proteins. Our study overall suggests that uc.8+ localization can be used as a prognostic biomarker for bladder cancer. ABSTRACT: Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.