Synthetic circular RNA functions as a miR-21 sponge to suppress gastric carcinoma cell proliferation

MicroRNA (miRNA) sponges containing miRNA complementary binding sites constitute a potentially useful strategy for miRNA-inhibition therapeutics in cancer patients. Recently, naturally occurring circular RNAs (circRNAs) have been revealed to function as efficient microRNA sponges. We hypothesized that synthetic circRNA sponges targeting oncomiRs could be constructed and used to achieve potentially therapeutic microRNA loss of function. In this study, linear RNA molecules containing five miR-21 binding sites were transcribed in vitro. After dephosphorylation by calf intestinal phosphatase and phosphorylation by T4 polynucleotide kinase, circRNA sponges were circularized using 5’-3’ end ligation by T4 RNA ligase 1. Synthetic circular sponge stability was assayed in the presence of RNase R or fetal bovine serum. Luciferase reporter and cell proliferation assays were performed to assess competitive inhibition of miR-21 activity by circRNA sponges in NCI-N87 gastric cancer cells. Tandem Mass Tag (TMT) labeling proteomics analysis and Western blotting were performed to delineate effects of circRNA sponges on miR-21 downstream targeted proteins. Our experiments revealed that artificial circRNA sponges can be synthesized using enzymatic ligation. These synthetic circRNA sponges are more resistant than their linear RNA counterparts to nuclease degradation in vitro. They effectively suppress the activity of miR-21 on its downstream protein targets, including the important cancer protein DAXX. Finally, they also inhibit gastric cancer cell proliferation. Our results suggest that synthetic circRNA sponges represent a rapid, effective, convenient strategy to achieve loss of miRNA function in vitro, with potential future therapeutic application in vivo.

携带微小RNA（microRNA, miRNA）互补结合位点的微小RNA海绵，是癌症患者开展miRNA抑制治疗的潜在有效策略。近年来，研究发现天然存在的环状RNA（circular RNAs, circRNAs）可作为高效的微小RNA海绵。本研究假设，靶向致癌miRNA（oncomiR）的人工合成circRNA海绵可被构建，并用于实现具有治疗潜力的miRNA功能缺失。本研究中，我们体外转录了携带5个miR-21结合位点的线性RNA分子。经小牛肠磷酸酶去磷酸化、T4多核苷酸激酶磷酸化处理后，我们利用T4 RNA连接酶1通过5’-3’末端连接实现了circRNA海绵的环化。我们在核糖核酸酶R（RNase R）或胎牛血清存在的条件下，检测了合成环状海绵的稳定性。在NCI-N87胃癌细胞中开展荧光素酶报告基因实验与细胞增殖实验，以评估circRNA海绵对miR-21活性的竞争性抑制作用。通过串联质量标签（Tandem Mass Tag, TMT）标记蛋白质组学分析与蛋白质免疫印迹（Western blotting）实验，我们阐明了circRNA海绵对miR-21下游靶蛋白的调控效应。本研究证实，通过酶促连接法可合成人工circRNA海绵。相较于对应的线性RNA分子，此类合成circRNA海绵在体外对核酸酶降解具有更强的抗性。它们可有效抑制miR-21对下游靶蛋白的调控活性，其中包括重要的癌症相关蛋白死亡结构域相关蛋白（DAXX）。最终，此类海绵还可抑制胃癌细胞的增殖。本研究结果表明，合成circRNA海绵是一种在体外实现miRNA功能缺失的快速、高效、便捷的策略，未来在体内治疗中具备潜在应用价值。