Figure 7.

SMAD4 is a direct target of miR-224. (a) Schematic diagram showing the matched sequences between the seed sequence of miR-224 and the 3′-UTRs of SMAD4. The lower case alphabets indicate the mutated nucleotides. (b) Luciferase reporter constructs containing WT or mutated SMAD4 3′-UTRs were co-transfected with pre-miR-224 or miR-224 sponge into 293T cells (n=4, mean±S.D., *P<0.01). (c) miR-224 represses SMAD4 protein level. Cells were transfected with NC or miR-224 mimic or infected with empty vector or pre-miR-224. Cell lysates were subjected to immunoblotting. miR-224 inhibition increases SMAD4 protein level. Cells were transfected with NC or miR-224 inhibitor or infected with empty vector or miR-224 sponge, and analyzed for SMAD4 protein level. (d) Knockdown or overexpression of miR-224 does not influence SMAD4 mRNA level. Total RNA was extracted from U2OS and MG-63 cells transfected with the indicated plasmids, and SMAD4 mRNA levels were determined by qRT-PCR (n=4, mean±S.D.)