Figure 5. miR-451 directly targets YWHAZ and subsequently suppresses YWHAZ-AKT signaling in AML cells.

A. The predicated potential binding site of miR-451 on the 3'UTR of YWHAZ. The “seed sequence” of miR-451 is marked with red color. B. Enforced miR-451 expression inhibited relative luciferase activity of the construct containing the wild type YWHAZ 3'UTR, but not the mutant. The dual-luciferase reporter assay was performed in triplicate in HEK-293 cells. Error bars represent SD (n=3); **P<0.01; Student's t-test. C. Overexpression of miR-451 reduced YWHAZ mRNA level in NB4 and HL-60 cells. **P<0.01. D. Enforced miR-451 expression reduced YWHAZ and subsequently decreased p-AKT protein levels in NB4 and HL-60 cells. E. knockdown of YWHAZ promoted apoptosis of NB4 and HL-60 cells. A representative experiment is presented on the left and a statistical analysis of three independent experiments on the right. **P<0.01. F. Knockdown of YWHAZ restrains proliferation of NB4 and HL-60 cells. G. Either knockdown of YWHAZ or knockdown of c-Myc inhibited activity of YWHAZ/AKT signaling in the AML cells. H. c-Myc inhibitor (JQ-1) treatment accelerated apoptosis in NB4 AML cells. A representative experiment is presented on the left and a statistical analysis of three independent experiments on the right. ***P<0.001. I. Immunoblotting of YWHAZ in the NB4 cells that were transfected with anti-451 (or anti-Con) for 24 h and then treated for another 48 h with si-YWHAZ (or si-Con). J. The cell apoptosis inhibition by anti-451 transfection can be rescued by re-transfection with si-YWHAZ. A representative experiment is presented on the left and a statistical analysis of three independent experiments on the right. *P<0.05; **P<0.01.