Department of Urology, Charité - University Medicine, 10117 Berlin, Germany.
Berlin Institute for Urologic Research, 10115 Berlin, Germany.
Theranostics. 2020 Jul 23;10(20):9268-9279. doi: 10.7150/thno.46341. eCollection 2020.
Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions. Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs (, and ), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6. The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6. Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies.
环状 RNA（circRNAs）是一类具有医学意义的新型 RNA。与线性 mRNA 转录本相比，circRNA 因其环状结构而具有更高的稳定性，这使其有望成为生物标志物。由于缺乏关于circRNA 稳定性的系统研究，这些研究取决于 RNA 完整性，以 RNA 完整性数（RIN）表示，在临床组织样本中，我们在本研究中在模型和临床条件下对此进行了研究。 从肾癌组织和细胞系（A-498 和 HEK-293）中提取的总 RNA 在热降解后具有不同的 RIN，用于模型实验。此外，还检查了在常规手术条件下收集的肾癌和前列腺癌组织中分离的 RNA，这些样本代表了 RIN 范围为 2 至 9 的临床样本。对几种 circRNAs（、和）、其相应的线性对应物、组织特异性参考基因和三个 microRNAs（作为对照）进行了定量实时逆转录聚合酶链反应（RT-qPCR）分析。使用线性回归分析将定量循环转化为相对数量，并将其归一化为相应组织中特定参考基因的表达。根据 RIN 将临床样本分为大于或小于 6 的两组。 模型实验和临床样本分析的结果表明，随着 RIN 值的降低，所有相对 circRNA 表达逐渐降低。在对数据进行归一化并将样本限制在 RIN 值>6 的样本后，RIN 的不利影响得到部分补偿。 我们的结果表明，circRNAs 在临床组织样本中不稳定，但受到与 mRNAs 相似的降解过程的影响。这在 circRNA 表达研究中尚未得到广泛研究，因此在获得可靠的 circRNA 表达数据时必须加以考虑。这可以通过应用在 mRNA 表达研究中常用的原则来实现。
