Fig. 5

circCPE serves as a miR-138 sponge. (A) The proposed circCP binding site on miR-138 was identified using RNAhybrid. (B) The miR-138 mimic was co-transfected with pCK-circCPE into HEK293T cells. Luciferase activities were measured 24 h after transfection. Renilla luciferase activity was normalized to Firefly luciferase activity. (C) The miR-138 biosensor (psiCHECK2-miR-34a 2×) was co-transfected with the miR-138 mimic and 1× or 2× pcD2.1-circCPE into HEK293T cells, and luciferase activities were measured after transfection. (D) The illustration shows the construct of circCPE containing wild-type miR-138 binding sites or mutated sites and schematic diagram of the miR-138 sensor structure. (E) miR-138 mimics were co-transfected with psiCHECK2-circCPE-WT (pCK-circCPE) or psiCHECK2-circCPE-MUT (pCK-circCPE-mut) into HEK293T cells, and luciferase activities were measured after transfection. (F) Ago2-RIP assay for the amount of circCPE and miR-138 in bovine myoblasts. RIP experiments showed that the anti-AGO2 antibody efficiently captured circCPE and miR-138 transcripts. (G) Effects of overexpression with circCPE on the expression level of miR-138. Values are means ±SEM for three individuals. P < 0.05, P < 0.01