Figure 1
Oncogenic Ras induced miR-134 expression via the ERK/JNK-AP-1 signaling pathways. (a) Heat map representation of the top 15 upregulated miRNAs in T29H cells compared with T29 cells. Rows, miRNAs; columns, profiled samples. (b) Quantification of six miRNAs in T29 and T29H cells using real-time RT-PCR. (c) Representative western blot images of H-Ras, p-ERK, ERK, p-JNK and Fra-1. GAPDH was used as loading control. (d) Quantification of AP-1 subunit levels in T29 and T29H cells using real-time RT-PCR. (e) Fra-1 knockdown inhibits AP-1 activity in T29H cells. (f) Diagram of the 4-kb region upstream of pre-miR-134. The AP-1 (gray and black box) and Fra-1 (black box) binding sites, the amplicons (F1–F6) utilized for ChIP PCR, and the DNA fragments incorporated into luciferase reporter constructs (P1–P3) were indicated. (g) ChIP assays with anti-Fra-1 in T29H and T29 cells. (h) Luciferase reporter assays with the various reporter constructs in T29 and T29H cells. (i) Fra-1 knockdown reduces miR-134 and pri-miR-134 levels in T29H cells. Fra-1 protein levels (top) and the relative RNA expression levels of Fra-1, miR-134 and pri-miR-134 (bottom). (j) The effect of MEK1/2 inhibitor U0126 on the miR-134 and pri-miR-134 abundance in T29H cells. The protein levels of ERK, p-ERK and Fra-1 (left) and the relative RNA levels of Fra-1, miR-134 and pri-miR-134 (right). (k) The protein levels of Fra-1 (left) and the relative RNA expression levels of Fra-1, miR-134 and pri-miR-134 (right) in T29H cells treated with the JNK inhibitor SP600125. Data are shown as mean±S.D. from three independent experiments. *P<0.05, **P<0.01 by Student’s t-test
