Figure 4.

Verification of NFATc1 as a target of miR-124 regulation. (A) The 3’-UTR region of NFATc1 is shown with the miR-124 target sequence highlighted. Two mutations were made to the NFATc1 3’-UTR to disrupt the putative miR-124 target sequence for use as controls in a luciferase assay. C3H10T1/2 cells grown in pellets under hypoxic conditions were transfected with miR-124 mimics or negative control (N.C.) sequences. The expression of NFATc1 was assessed at the mRNA level by RT-PCR (B) and the protein level by Western blot (C). The expression of chondrogenesis marker genes was assessed by RT-PCR assays in cells transfected with either the miR-124 mimic or the N.C. sequence at the time points indicated. (D) Sox9; (E) aggrecan; (F) collagen IIa1; (G) collagen Xa1. (H) Dual luciferase reporter assays were performed in HEK293 cells to test whether there was an interaction between miR-124 and the wildtype NFATc1 3’-UTR or either mutant NFATc1 3’-UTR. (I) The miR-124 inhibitor increased expression of NFATc1 at the protein level. MiR-124 effects were analyzed in parallel as a positive control. (pGL3-Rmut1, pGL3-Rmut2). The bars represent means ± SD (n=3). *P<0.05.