Figure 3.

miR-224 is a direct target gene of TAZ-TEAD. (a) TAZ-regulated miRNAs. miRNAs regulated by TAZ were identified by miRNA high-throughput sequencing. miRNAs with a P-value of <0.01 in TAZ knockdown MG-63 cells were chosen, and the heat map was drawn using Matlab. (b and c) miR-224(b) and pri-miR-224 (c) were highly expressed in OS cells. Quantitative PCR (qPCR) analysis was used to quantify the endogenous levels of miR-224 and pri-miR-224 in OS cells. U6 and β-actin were used as controls. (d and e) TAZ induces miR-224 expression. The miR-224 expression levels in U2OS and MG-63 stable cells with TAZ overexpression (d) or TAZ knockdown (e) were determined by quantitative reverse transcription (qRT-PCR). Experiments were performed in triplicate. (f) TAZ binds to the promoter of miR-224. ChIP was performed using anti-FLAG or anti-IgG antibodies, followed by qPCR using primers specific to the indicated promoter regions (n=4, mean±S.D., *P<0.01). Data were obtained from three independent experiments. (g) Lats1/2 knockdown induces miR-224 expression. The miR-224 expression levels in U2OS and MG-63 stable cells with Lats1/2 knockdown were determined by qRT-PCR. Experiments were performed in triplicate. (h) Knockdown of TEADs reduces miR-224 level. Cells were transfected with shRNA targeting TEAD1/3/4. The levels of mature miR-224 were determined. Experiments were performed in triplicate (n=4, mean±S.D., *P<0.01). (i) The miR-224 promoter region contains two putative TEAD-binding sites. The putative TEAD-binding sites (TB1–TB2) are shown in the frame. (j) The putative TEAD-binding sites are important for miR-224 promoter activity. The putative TEAD-binding sites (TB) were mutated individually or in combination. The luciferase activity of each reporter was measured in the presence or absence of TAZ. The activation folds by TAZ are shown (n=4, mean±S.D., *P<0.01 **P<0.001)