Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, 518039, China; Shantou University Medical College, Shantou, 515041, Guangdong Province, China; Department of Urology, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, 518039, China.
Key Laboratory of Medical Reprogramming Technology, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, 518039, China; Department of Urology, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, 518039, China.
Cancer Lett. 2018 May 28;422:94-106. doi: 10.1016/j.canlet.2018.02.038. Epub 2018 Mar 1.
Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play important roles in human cancers, acting as transcriptional and post-transcriptional regulators, respectively. These phenomena raise questions about the ability of an artificial device to simultaneously regulate miRNAs and TFs. In this study, we aimed to construct artificial long non-coding RNAs, "alncRNAs", and to investigate their therapeutic effects on bladder cancer cell lines. Based on engineering principles of synthetic biology, we combined tandem arrayed aptamer cDNA sequences for TFs with tandem arrayed cDNA copies of binding sites for the miRNAs to construct alncRNAs. In order to prove the utility of this platform, we chose β-catenin and the miR-183-182-96 cluster as the functional targets and used the bladder cancer cell lines 5637 and SW780 as the test models. Dual-luciferase reporter assay, real-time quantitative PCR (qRT-PCR) and related phenotypic experiments were used to test the expression of related genes and the therapeutic effects of our devices. The result of dual-luciferase reporter assay and qRT-PCR showed that alncRNAs could inhibit transcriptional activity of TFs and expression of corresponding microRNAs. Using functional experiments, we observed decreased cell proliferation, increased apoptosis, and motility inhibition in alncRNA-infected bladder cancer cells. What's more, follow-up mechanism experiments further confirmed the anti-tumor effect of our devices. In summary, our synthetic devices indeed function as anti-tumor regulators, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cells. Most importantly, anti-cancer effects were induced by the synthetic alncRNAs in the bladder cancer lines. Our devices, all in all, provided a novel strategy and methodology for cancer studies, and might show a great potential for cancer therapy if the challenges of in vivo DNA delivery are overcome.
致癌转录因子 (TFs) 和 microRNAs (miRNAs) 在人类癌症中均发挥重要作用，分别作为转录和转录后调控因子。这些现象引发了人们对于一种人工装置是否能够同时调控 miRNAs 和 TFs 的疑问。在本研究中，我们旨在构建人工长非编码 RNA（alncRNA），并研究其对膀胱癌细胞系的治疗效果。基于合成生物学的工程原理，我们将 TF 的串联排列适体 cDNA 序列与 miRNA 结合位点的串联排列 cDNA 拷贝结合起来构建 alncRNA。为了证明该平台的实用性，我们选择β-catenin 和 miR-183-182-96 簇作为功能靶标，并使用膀胱癌细胞系 5637 和 SW780 作为测试模型。双荧光素酶报告基因检测、实时定量 PCR（qRT-PCR）和相关表型实验用于检测相关基因的表达和我们装置的治疗效果。双荧光素酶报告基因检测和 qRT-PCR 的结果表明，alncRNA 可以抑制 TF 的转录活性和相应 miRNA 的表达。通过功能实验，我们观察到 alncRNA 感染的膀胱癌细胞增殖减少、凋亡增加和运动抑制。此外，后续的机制实验进一步证实了我们装置的抗肿瘤作用。总之，我们的合成装置确实可以作为抗肿瘤调节剂，在膀胱癌细胞中同步完成转录和转录后调控。更重要的是，合成 alncRNA 在膀胱癌系中诱导了抗癌作用。总的来说，我们的装置为癌症研究提供了一种新的策略和方法，如果克服了体内 DNA 递送的挑战，它们可能为癌症治疗带来巨大潜力。
