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.