Institut für Molekulare Medizin, Universität zu Lübeck and UKSH, Campus Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany.
Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
Sci Rep. 2021 Nov 11;11(1):22114. doi: 10.1038/s41598-021-01548-0.
Numerous indirect and in silico produced evidences suggest circular RNAs (circRNA) in mammals while thorough experimental proofs of their existence have rarely been reported. Biological studies of circRNA, however, should be based on experimentally verified circRNAs. Here, we describe the identification of two circRNAs originating from the gene locus of the translocation associated membrane protein 1 (TRAM1). Linear and potentially circular TRAM1-specific transcripts were identified in a transcriptome analysis of urine RNA of bladder cancer (BCa) patients versus healthy donors. Thus, we first focused on the topology of TRAM1-specific transcripts. We describe conclusive experimental evidence for the existence of TRAM1-specific circRNAs in the human BCa cell lines ECV-304 and RT-4. PCR-based methodology followed by cloning and sequencing strongly indicated the circular topology of two TRAM1 RNAs. Further, studies with exon fusion sequence-specific antisense oligonucleotides (asON) and RNase H as well as studies in the use of RNase R contribute to conclusive set of experiments supporting the circular topology of TRAM1 transcripts. On the biological side, TRAM1-specific circRNAs showed low expression levels and minor differences in BCa cell lines while linear TRAM1 transcripts displayed down-regulated expression in the higher cancer stage model ECV-304 versus more differentiated RT-4 cells.
大量间接和计算机模拟产生的证据表明哺乳动物中存在环状 RNA（circRNA），但很少有关于其存在的彻底实验证据。然而，circRNA 的生物学研究应该基于经过实验验证的 circRNA。在这里，我们描述了两种源自易位相关膜蛋白 1（TRAM1）基因座的 circRNA 的鉴定。在膀胱癌（BCa）患者与健康供体的尿 RNA 转录组分析中，鉴定出线性和潜在的环状 TRAM1 特异性转录本。因此，我们首先专注于 TRAM1 特异性转录本的拓扑结构。我们描述了在人类 BCa 细胞系 ECV-304 和 RT-4 中存在 TRAM1 特异性 circRNA 的确凿实验证据。基于 PCR 的方法，随后进行克隆和测序，强烈表明两种 TRAM1 RNA 的环状拓扑结构。此外，使用exon 融合序列特异性反义寡核苷酸（asON）和 RNase H 以及在使用 RNase R 方面的研究，为支持 TRAM1 转录物环状拓扑结构的一系列确凿实验做出了贡献。在生物学方面，TRAM1 特异性 circRNA 在 BCa 细胞系中的表达水平较低且差异较小，而线性 TRAM1 转录本在更高的癌症阶段模型 ECV-304 中表达下调，而在分化程度更高的 RT-4 细胞中表达下调。
