Figure 3 Down-regulation of WT1 by siRNA could not increase the expression of miR-15a/16-1 in leukemic cells. (A and B) K562 and HL-60 cells were transfected with 50 nM siRNA-WT1, 50 nM N.C or neither of the above for 24 and 48 hours, then the relative mRNA expression of WT1 and the corresponding WT1 protein were respectively measured by quantitative real-time PCR and Western blotting. GAPDH as loading control. (C and D) The relative expressions of Selleckchem NVP-LDE225 miR-15a and miR-16-1 were measured by qRT-PCR after K562 and HL-60 cells were

transfected with 50 nM siRNA-WT1, 50 nM N.C or neither of the above for 24 and 48 hours. * and & P < 0.01 versus negative control (N.C). Anti-miR-15a/16-1 oligonucleotides (AMO) partly reversed the down-regulation of WT1 induced by curcumin in leukemic cells To further confirm that pure curcumin down-regulated the expression of WT1 by up-regulation Proteasomal inhibitor of miR-15a/16-1, 20 uM curcumin treated-K562 JNK-IN-8 and 10

uM curcumin treated- HL-60 cells were transfected with 50 nM anti-miR-15a/16-1 oligonucleotides for 48 hours. The levels of WT1 protein were detected by Western blotting after transfection. As Figure 4A and 4B demonstrated that anti-miR-15a/16-1 oligonucleotides could effectively decrease the expression of miR-15a and miR-16-1 in K562 and HL-60 cells. Moreover, anti-miR-15a/16-1 oligonucleotides partly abolished the inhibitory effect of curcumin on WT1 protein expression (Figure 4C and 4D). Finally, as Demeclocycline indicated in Figure 4E and 4F, 20 uM curcumin treated-K562 and 10 uM curcumin treated-HL-60 cells were transfected with 50 nM of anti-miR-15a/16-1 oligonucleotides

for 24, 48 and 72 hours, the CCK-8 assay revealed that anti-miR-15a/16-1 oligonucleotides effectively reversed the inhibition of cell proliferation caused by curcumin in K562 and HL-60 cells. Figure 4 Anti-miR-15a/16-1 oligonucleotides (AMO) partly reversed the downregulation of WT1 induced by curcumin in K562 and HL-60 cells. (A and B) The relative expressions of miR-15a/16-1 were measured by qRT-PCR after K562 and HL-60 cells were transfected with 50 nM of anti-miR-15a/16-1 oligonucleotides for 48 hours. * and & P < 0.01 versus negative control (SCR). (C and D) 20 uM curcumin treated-K562 and 10 uM curcumin treated- HL-60 cells were transfected with 50 nM of anti-miR-15a/16-1 oligonucleotides for 48 hours, then the protein levels of WT1 were measured by Western blotting. GAPDH as loading control. (E and F) 20 uM curcumin treated-K562 and 10 uM curcumin treated- HL-60 cells were transfected with 50 nM of anti-miR-15a/16-1 oligonucleotides for 24, 48, and 72 hours, then cell proliferation was measured by CCK-8 assay. # and $ represent less than 0.05 of p-values, compared respectively with pure curcumin treatment alone at the same time.