showed that WT1 was a target of miR-15a/16-1 in MEG-01 cells by microarray and proteomics analysis[10]. However whether WT1 was directly targeted by miR-15a/16-1 in K562 and HL-60 cells was not verified in lab. As indicated in Figure 2A, over-expression of miR-15a/16-1

in K562 and HL-60 cells obviously reduced the protein level of WT1 at 24 and 48 h after transfection with pRS-15/16 compared with normal controls, whereas the level of WT1 mRNA was not significantly affected (Figure 2B). Then we cloned the 3′UTR region of WT1 downstream of a luciferase reporter gene and corresponding negative control into K562 and HL-60 cells, but the luciferase activity Selleckchem Smoothened Agonist of cells transfected with pRS-15/16 was not significantly decreased compared with the negative control (Figure 2C and 2D). Bcl-2 is a target of posttranscriptional repression by miR-15 and miR-16-1, which act as a positive control[9]. Figure 2 miR-15a/16-1 downregulates WT1 protein level not through BGB324 targeting mRNAs according to the degree of complementarity with their 3′UTR.

(A) K562 and HL-60 cells were transiently transfected with pRS-15/16 or pRS-E vector for different time periods and subjected to western analysis with the indicated antibodies. The level of GAPDH was used as a loading control. (B) K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E vector for 24 and 48 hours, then the relative expression of WT1 was measured by quantitative real-time PCR. (C and D). K562 and HL-60 cells were transfected with the pGL-3 containing Bcl-2 3′UTR or PI-1840 WT1 3′UTR and pRS-15/16 or pRS-E for 24 hours, relative repression fold of firefly luciferase expression was standardized to Renilla luciferase, pGL-TK. Anti-miR-15a/16-1 oligonucleotides (AMO) reversed the expression of WT1 in K562 and HL-60 cells In order to investigate the effect of AMO-miR-15a/16-1 on WT1 expression, we transfected AMO-miR-15a/16-1 to K562 and HL-60 cells for 24 and 48 h. miR-15a/16-1 and U6 snRNA expression was determined by quantitative real-time PCR. U6 snRNAs were used as the internal control. The fold-change for

miR-15a/16-1 expression level was calculated using ΔCT and 2-ΔΔCT, as described in the Materials and methods. As indicated in Figure 3A and 3B, AMO effectively decreased the expression of miR-15a/16-1 in K562 and HL-60 cells. Meanwhile the protein level of WT1 was increased but the mRNA level of WT1 was not affected by AMO-miR-15a/16-1 at 48 hours compared with control group (SCR) in K562 and HL-60 cells (Figure 3C and 3D). Figure 3 AMO-miR-15a/16-1 reversed the expression of WT1 in K562 and HL-60 cells (A) and (B) AMO inhibited the expression of miR-15a/16-1. K562 and HL-60 cells were incubated with AMO-miR-15a/16-1 for 24 and 48 hours, then miR-15a/16-1 and U6 snRNA expression were determined by quantitative real-time PCR.