Figure 3.

Mir-204 and miR-211 bind to the 3′-UTR of the Runx2 gene. (A): Clonal C3H10T1/2 cells, in which Runx2 is overexpressed under the control of the CMV promoter and SV40 3′-UTR (Runx2 3′-UTR deletion), were infected with control (empty vector), miR-204, or miR-211 expressing retrovirus. After 15-day selection with puromycin, cells were harvested for Runx2 protein expression using western blot analysis. Overexpression of miR-204 or miR-211 did not change Runx2 protein levels, suggesting that Runx2 3′-UTR may be the target of miR-204/211. (B): A diagram of Runx2 3′-UTR reporters. m1, m2, m3, m4, and m5 denote five different Runx2 3′-UTR reporters, which contain one mutated site that cannot be bound to miR-204. (C): The wild-type (WT) Runx2 3′-UTR reporter was cotransfected with miR-204 or anti-miR-204 oligos into C3H10T1/2 cells. Forty-eight hours after transfection, luciferase activities were measured (n = 6). (D): The wild-type Runx2 3′-UTR reporter or mutated 3′-UTR reporter was cotransfected with miR-204 or control oligos into C3H10T1/2 cells. Forty-eight hours after transfection, luciferase activities were measured. *, p < .05, compared with WT for mutated reporters, ANOVA followed by Dunnett’s test.