MicroRNA (miR) sponges containing miR binding sequences constitute a potentially powerful molecular therapeutic strategy. Recently, naturally occurring circular RNAs (circRNAs) were shown to function as efficient miR sponges in cancer cells. We hypothesized that synthetic circRNA sponges could achieve therapeutic loss-of-function targeted against specific miRs. Linear RNA molecules containing miR-21 binding sites were transcribed in vitro; after dephosphorylation and phosphorylation, circularization was achieved using 5′-3′ end-ligation by T4 RNA ligase 1. circRNA stability was assessed using RNase R and fetal bovine serum. Competitive inhibition of miR-21 activity by a synthetic circRNA sponge was assessed using luciferase reporter, cell proliferation, and cell apoptosis assays in three gastric cancer cell lines. circRNA effects on downstream proteins were also delineated by Tandem Mass Tag (TMT) labeling (data available via ProteomeXchange identifier PRIDE: PXD008584), followed by western blotting. We conclude that artificial circRNA sponges resistant to nuclease digestion can be synthesized using simple enzymatic ligation steps. These sponges inhibit cancer cell proliferation and suppress the activity of miR-21 on downstream protein targets, including the cancer protein DAXX. In summary, synthetic circRNA sponges represent a simple, effective, convenient strategy for achieving targeted loss of miR function in vitro, with potential future therapeutic application in human patients.

含有 miR 结合序列的 MicroRNA (miR) 海绵构成了一种潜在强大的分子治疗策略。最近，天然存在的环状 RNA（circRNA）被证明在癌细胞中可作为有效的 miR 海绵。我们假设合成的 circRNA 海绵可以实现针对特定 miR 的功能丧失治疗。含有miR-21结合位点的线性RNA分子在体外转录；去磷酸化和磷酸化后，通过 T4 RNA 连接酶 1 使用 5'-3' 末端连接实现环化。使用 RNase R 和胎牛血清评估 circRNA 稳定性。在三种胃癌细胞系中，使用荧光素酶报告基因、细胞增殖和细胞凋亡测定来评估合成 circRNA 海绵对 miR-21 活性的竞争性抑制。 circRNA 对下游蛋白质的影响也通过串联质量标签 (TMT) 标记（数据可通过 ProteomeXchange 标识符 PRIDE：PXD008584 获得）描述，然后进行蛋白质印迹。我们得出的结论是，可以使用简单的酶连接步骤合成抗核酸酶消化的人工 circRNA 海绵。这些海绵抑制癌细胞增殖并抑制 miR-21 对下游蛋白靶标（包括癌症蛋白 DAXX）的活性。总之，合成的 circRNA 海绵代表了一种简单、有效、方便的策略，可在体外实现 miR 功能的靶向丧失，并具有潜在的未来在人类患者中的治疗应用。