Figure 2. miR21 upregulates D3 expression by targeting GRHL3.

(A) BCC cells were transiently transfected with miR21 expression plasmid, and cells were collected at the indicated times. D3 mRNA and protein were measured by real-time PCR and Western blot (see complete unedited blots in the supplemental material). (B) LNA anti-miR21 or a control LNA-scrambled oligo was transfected in BCC cells, and D3 mRNA and protein were measured as in A. (C) BCC cells transfected with the artificial T3-responsive promoter TRE3-TK-Luc and CMV-renilla as internal control. Increasing amounts of miR21 were cotransfected, and cells were analyzed for luciferase activity. The results are shown as means ± SD of the luciferase/renilla (LUC/Renilla) ratios from at least 3 separate experiments, performed in duplicate. (D) miR21 target genes were classified for functional classification with the DAVID program (36). (E) D3 protein levels were measured by Western blot in BCC cells transfected with Flag-GRHL3 and harvested at the indicated times. GRHL3 (anti-Flag) and tubulin levels were measured as control. (F) Primary cultures of keratinocytes were transfected with miR21, and cells were harvested at 48 and 72 hours. Dio3 and Grhl3 mRNAs were measured by real-time PCR. (G) BCC cells were transfected with 2 different siRNAs targeting endogenous Grhl3 oligos or a combination of them, and D3 protein was measured by Western blot analysis. (H) Schematic representation of the Dio3 locus, the Dio3 promoter, and the G1 site (red) responsive to GRHL. (I and J) BCC cells were transiently transfected with Dio3-Luc promoter (I) or Dio3-Luc and mutDio3-Luc promoters (J) and with increasing amounts of the GRHL3 plasmid. Cells were harvested 48 hours after transfection and analyzed for luciferase activity. CMV-renilla was cotransfected as internal control. The results are shown as means ± SD of the LUC/renilla ratios from at least 3 separate experiments, performed in duplicate. *P < 0.05.