miR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level

Abstract

Background: miR-15a and miR-16-1(miR-15a/16-1) have been implicated as tumor suppressors in chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemic cells. However the mechanism of inhibiting the proliferation of leukemic cells is poorly understood.

Methods: K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E, cell growth were measured by CCK-8 assay and direct cell count. Meanwhile WT1 protein and mRNA level were measured by Western blotting and quantitative real-time PCR.

Results: In this study we found that over-expression of miR-15a/16-1 significantly inhibited K562 and HL-60 cells proliferation. Enforced expression of miR-15a/16-1 in K562 and HL-60 cells significantly reduced the protein level of WT1 but not affected the mRNA level. However enforced expression of miR-15a/16-1 can not reduce the activity of a luciferase reporter carrying the 3'-untranslated region(3'UTR) of WT1. Silencing of WT1 by specific siRNA suppressed leukemic cells proliferation resembling that of miR-15a/16-1 over-expression. Anti-miR-15a/16-1 oligonucleotides (AMO) reversed the expression of WT1 in K562 and HL-60 cells. Finally, we found a significant inverse correlation between miR-15a or miR-16-1 expression and WT1 protein levels in primary acute myeloid leukemia (AML) blasts and normal controls.

Conclusions: These data suggest that miR-15a/16-1 may function as a tumor suppressor to regulate leukemic cell proliferation potentially by down-regulating the WT1 oncogene. However WT1 is not directly targeted by miR-15a/16-1 through miRNA-mRNA base pairing, therefore more study are required to understand the mechanism by which miR-15a/16-1 downregulate WT1.

Figure 1

Effects of miR-15a/16-1 on the proliferation of K562 and HL-60 cells. K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E vector (negative control) for 24, 48 and 72 hours, then the relative expressions of miR-15a/16-1 were measured by qRT-PCR (A and B). CCK-8 assay (C and E) and direct cell count (D and F) were performed when K562 and HL-60 cells were transfected with pRS-15/16 or pRS-E vector at different time periods. Data are shown as mean ± SD from three independent experiments. *P < 0.05 versus negative control.

Figure 2

miR-15a/16-1 downregulates WT1 protein level not through 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 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.

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. (C) and (D) K562 and HL-60 cells were incubated with AMO-miR-15a/16-1 for 48 h, then mRNA and protein level of WT1 were detected by quantitative real-time PCR and Western blotting individually. *P < 0.01 versus SCR.

Figure 4

The role of miR-15a/16-1 in the regulation of leukemic cell proliferation. (A) and (B) K562 and HL-60 cells were incubated with 1.5 ug siRNA-WT1, 1.5 ug N.C or neither of the above for 24 and 48 hours, then the relative expression of WT1 and the corresponding WT1 protein level were separately measured by quantitative real-time PCR and Western blotting. (C) and (D) K562 and HL-60 cells treated with siRNA or N.C or neither of the above were measured by CCK-8 assay at different time periods. Data are shown as mean ± SD from three independent experiments. *P < 0.05 versus negative control.

Figure 5

WT1 protein expression is inversely correlated with miR-15a or miR-16-1 expression in AML samples and normal controls. (A) WT1 protein levels from 2 normal controls (N1 and N2) and 6 AML samples (P1-P6) were measured by Western blotting. The numbers represent the relative expression of miR-15a and miR-16-1 in the same specimens. (B) and (C) Inverse correlation between miR-15a or miR-16-1 expression and WT1 protein level in 25 primary AML samples and 5 normal controls. A statistically significant correlation between miR-15a or miR-16-1 expression and WT1 protein level was observed by Pearson's method. WT1 verse miR-15a R = -0.73 P < 0.01; WT1 verse miR-16-1 R = -0.76 P < 0.01