Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation

Abstract

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.

Integrated analysis of circular RNA‑associated ceRNA network in pancreatic ductal adenocarcinoma

“…These characteristics make circRNAs potential biomarkers for several types of tumor (28,29). Although the exact functions of the majority of circRNAs remain unclear, a number of recent studies have suggested that circRNAs affect the initiation and development of different types of malignancies (30)(31)(32). Cao et al (32) reported that circRNA_100876 was highly expressed in esophageal squamous cell carcinoma.…”

Novel circular RNA hsa_circ_0077837 suppresses colorectal cancer cell proliferation by regulating the microRNA 21/phosphatase and tensin homolog axis

“…In our study, we further confirmed that miR-21 expression in CRC suppressed the Pten/AKT pathway and promoted CRC cell proliferation. Recently, linear sponge RNAs including miR sites were confirmed to cause miR-21 function loss [29,30], which was considered as the basis of a promising strategy to suppress miR-21 expression. In our study, we confirmed that circEPB41L2 was dramatically increased and could sponge miR-21 to suppress CRC proliferation.…”

The Regulatory Functions of Circular RNAs in Digestive System Cancers

“…CircRNAs can act as sponges for miRNAs. Artificial circRNA sponges resistant to nuclease digestion could be synthesised using simple enzymatic ligation steps [108]. The synthetic circular miR-21 (scRNA21) sponge inhibited cancer cell proliferation and suppressed the activity of miR-21 on downstream protein targets, including the cancer protein DAXX [108].…”

“…Artificial circRNA sponges resistant to nuclease digestion could be synthesised using simple enzymatic ligation steps [108]. The synthetic circular miR-21 (scRNA21) sponge inhibited cancer cell proliferation and suppressed the activity of miR-21 on downstream protein targets, including the cancer protein DAXX [108]. Therefore, by studying the structure of endogenous circular RNA sponges to design and develop effective artificial sponges, we could eventually regulate the function of miRNAs in diseases.…”