Fig. 1

MiR-31-5p was able to interact with CamkIIδ directly. a The schematic diagram of luciferase reporter construct (pmir-GLO-CaMKIIδ-WT) and mutated reporter construct (pmir-GLO-CaMKIIδ-MUT). The sequences predicted by TargetScan for imperfect binding between mouse miR-31-5p (mmu-miR-31-5p) and 3′ UTR of CamkIIδ, and the vector sequences of WT (pmir-GLO-CaMKIIδ-WT) and mutant (pmir-GLO-CaMKIIδ-MUT) designed for luciferase activity assay. b, c Excess RA up-regulated miR-31-5p level and inhibited CamkIIδ expression in both mouse embryonic tongue and C2C12 cells. In vivo, the expression of miR-31-5p was up-regulated at E14.5 in the tongue of RA-treated mouse, nevertheless, the expression of CamkIIδdown-regulated at E14.5. In vitro, the relative expression of miR-31-5p was up-regulated and CamkIIδwas inhibited in the C2C12 myoblasts after 24 h supplemented with 10 μM RA. d Direct interaction between miR-31-5p and CamkIIδ. Plasmids of pmir-GLO-CaMKIIδ-WT, pmir-GLO-CaMKIIδ-MUT, and pmir-GLO were co-transfected into 293 T cells with duplex NC and miR-31-5p mimics. Luciferase activities were detected about 24 h after transfection. MiR-31-5p but not duplex NC, significantly inhibited the luciferase activity of the pmir-GLO-CaMKIIδ-WT; in contrast, it did not inhibit the activities of pmir-GLO-CaMKIIδ-MUT and pmir-GLO. (The difference time points in b and c were normalized by GAPDH expression as inner control; *P < 0.05, **P < 0.01; pmir-GLO in d was the empty vector working as inner control)