Figure 3: Prdm16 is a direct target of the miR-23a cluster and suppresses Sost.

(a) RNA-Seq with IPA and Targetscan analyses revealed that TGF-β is the most differentially regulated signalling pathway (P value=4.75E−06) and that Prdm16 is a possible repressive upstream regulator of TGF-β signalling (P value=1.47E−02). (b) The conserved seed sequence of miR-27a in the 3′UTR of Prdm16 mRNA among different species. (c) HEK293T cells were independently co-transfected with psiCheck-2 luciferase reporter containing 3′UTR Prdm16 along with the GFP control, miR-27a or miR-27a decoy (N=3 for each group). (d) MC3T3-E1 cells were transduced with miR-27a (27A)- or miR-27a decoy (27D)-expressing lentivirus. Twenty-four hours before transfection of the psiCheck-2 luciferase reporter containing 3′UTR Prdm16, cells were treated with 1 μM of doxycycline (Dox) or vehicle (control); 48 h after transfection of the reporter vectors, cells were collected for luminescence signal reading (N=4 for each group). (e) Expression levels of Sost in BMSCs from WT or Col1a1-miR-23aC (23aC) mice transduced with GFP- or Prdm16-expressing lentivirus on day 28 of differentiation (N=3 for each group). (f) Prdm16 and Smad3 are co-localized at the promoter region (including one SBE) from 1,036 to 1,328 bp upstream of the Sost transcription start site (TSS) in MC3T3-E1 cells. After 24 h of TGF-β treatment (10 ng ml−1), acetylation of histone H3 (H3Ac) was increased with the recruitment of Smad3. Upon induction of Prdm16 (1 μM of doxycycline), H3Ac levels were decreased at the Sost promoter region. CTL, control without Dox. This experiment was repeated three times. Statistical analyses used one-way ANOVA for multiple groups. Data are shown as mean±s.d.; *P<0.05, ***P<0.005 and ****P<0.001.