Figure 3
MiR-100 directly targets RBSP3, which responds to ATRA and 1,25D induction. (a) Sequences of the predicted miR-100 binding sites in human, mouse and rat genomic regions. Highly conserved nucleotides are shown in blue. (b) Schematic of the luciferase reporter assay used to validate the interaction between miR-100 and the 3′ UTR of RBSP3. Red font indicates the ‘seed’ regions. MRE RBSP3 3′ UTR of wild, deleted, point mutant and full mutant were separately inserted into a psiCheck2 vector downstream from the Renilla luciferase gene. (c) Repression of luciferase activity due to the interaction between miR-100 and the luciferase constructs. Each Renilla luciferase reading was normalized to that obtained for the control firefly luciferase. (d) Western blot analysis of the expression level of RBSP3 following the overexpression of miR-100 mimics or the knockdown of endogenous miR-100 using miR-100 antisense in HL60 cells. (e) Inverse correlation of expression levels of miR-100 and RBSP3 protein in AML patients (from AML-M1 to M5) demonstrated by qRT–PCR and western blot. (f, g) Time-course analysis of the induced upregulation of RBSP3 protein in HL60 cells by 1 μM ATRA and 100 nM 1,25D3, respectively. RBSP3 protein was gradually upregulated from 4 h to 24 h after treatment with 100 nM 1,25D3. (h) Dose-dependent effect of ATRA and 1,25D3 on RBSP3 protein expression at 96 h after treatments. RBSP3 protein expression increased in a dose-dependent manner following treatment with 0.1 uM/1 uM ATRA or 1 nM/100 nM 1,25D3. A full colour version of this figure is available at the Oncogene journal online.
