Figure 6.

Circular miR-21-5p RNA decoys impair cancer cell proliferation and invasion. (A) Schematic showing the synthesis strategy of ciRs containing four miRNA-binding or control sequences. Single, perfectly complementary and bulged miR-21-5p binding sites are depicted (right panel). (B) Representative northern blot analysis (n = 3 experiments) of linear and circular RNAs before (left) and upon RNA ligation (right). (C) Representative northern blot analysis of miR-21-5p associated with linear (top panel) or circular (bottom panel) immobilized miRNA decoys in transfected ES-2 cells. EB, empty beads. (D) Decay of linear (red) and circular (green) bulged miR-21-5p decoys was determined by northern blotting upon transfection in ES-2 cells. The biphasic degradation (circular + relinearized) of circular RNA decoys is shown in black. Half-lives of indicated RNA species are indicated (n = 3 experiments). (E) Proliferation of A549 or ES-2 spheroids was determined by using CellTiter Glo upon transfection with indicated ciRs in four experiments. Representative bright-field images of ES-2 spheroids (left panel). (F) Representative bright-field images of ES-2 spheroids invading Matrigel upon transfection of indicated ciRs (n = 5 experiments). The invasive fronts are shown in red (Ctrl) or blue (miR-21-5p decoys complementary and bulged). (G) RT-qPCR analysis of mRNA levels in A549 cells transfected with bulged miR-21-5p ciRs normalized to Ctrl ciRs. RPLP0 served as a normalization and EEF2 as a negative control in three experiments. Error bars indicate SD. Statistical significance, indicated by P-values, was determined by Student’s t-test.