Table_4_Back-Splicing Transcript Isoforms (Circular RNAs) Affect Biologically Relevant Pathways and Offer an Additional Layer of Information to Stratify NMIBC Patients.XLSX

Circularized transcript isoforms due to back-splicing are increasingly being reported in different tissues types and pathological states including cancer. Since these circular RNAs (circRNAs) are more stable than linear messenger RNA their identification and profiling in tumor tissue could aid in stratifying patients and may serve as biomarkers. In this study, we have investigated the relationship between circRNA expression and tumor grade in a cohort of 58, mostly non-muscle-invasive bladder cancer patients. From 4571 circRNAs detected, we identified 157 that were significantly differentially expressed between tumor grades relative to the linear transcript. We demonstrated that such grade-related differences can be identified in an independent cohort, and that a large fraction of circRNAs can be, in principle, detected in urine. The differentially expressed circRNAs cluster into subgroups according to their co-expression, subgroups which are enriched for DNA repair, cell cycle and intracellular signaling genes. Since one proposed function of circRNAs is to interfere with gene-regulation by acting as microRNA “sponges,” candidates which were differentially expressed between tumor grades were investigated for potential miRNA target sites. By investigating the circRNAs from bladder cancer related pathways we demonstrated that the expression of these pathways, the circRNAs, and their parental genes are often decoupled and do not correlate, yet that some circRNAs do not follow this tendency. The present study provides the next step for the comprehensive evaluation of this novel class of RNAs in the context of non-muscle-invasive bladder cancer. Intriguingly, despite their possible function as microRNA sponges, they potentially affect host mRNA levels at the transcriptional stage, as compared to post-transcriptional control by miRNAs. Our analysis indicates differences of their activity between bladder cancer tumor stages, and their relative expression levels may provide an additional layer of information for patient stratification.

由反向剪接产生的环状转录本异构体，目前已在包括癌症在内的多种组织类型与病理状态中被日益频繁地报道。这类环状RNA（circular RNAs, circRNAs）相较于线性信使RNA（linear messenger RNA, mRNA）更为稳定，因此在肿瘤组织中对其开展鉴定与表达谱分析，或可助力患者分层，并有望作为生物标志物（biomarker）应用于临床。本研究针对由58名患者（绝大多数为非肌层浸润性膀胱癌患者）组成的队列，探究了环状RNA表达水平与肿瘤分级之间的关联。在检测到的4571条环状RNA中，我们鉴定出157条相较于其对应的线性转录本，在不同肿瘤分级间呈现显著差异表达的环状RNA。研究证实，这类与肿瘤分级相关的表达差异可在独立队列中复现，且原则上可在尿液中检测到相当比例的环状RNA。差异表达的环状RNA可根据共表达模式聚类为不同亚组，这些亚组显著富集DNA修复、细胞周期及细胞内信号传导相关基因。鉴于环状RNA的一项已被提出的功能是作为微小RNA（microRNA, miRNA）“海绵”干扰基因调控，我们针对肿瘤分级间差异表达的候选环状RNA，对其潜在的miRNA结合位点进行了系统探究。通过聚焦膀胱癌相关通路中的环状RNA开展分析，我们发现这些通路对应的环状RNA及其母基因的表达水平往往相互解耦，不存在显著相关性，但部分环状RNA并不遵循这一规律。本研究为在非肌层浸润性膀胱癌场景下全面评估这类新型RNA提供了重要的研究进展。值得注意的是，尽管环状RNA可能具备微小RNA海绵的功能，但其对宿主信使RNA（messenger RNA, mRNA）水平的调控作用可能发生在转录阶段，而非通过miRNA实现的转录后调控。我们的分析显示，不同膀胱癌肿瘤分期中环状RNA的活性存在显著差异，其相对表达水平或可为患者分层提供额外的信息维度。