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. 2014 Nov 26;6(2):1249–1261. doi: 10.18632/oncotarget.2859

Figure 3. MiR-21 is directly and epigenetically downregulated by RBP2.

Figure 3

(A) miRNA expression analysis of the level of miR-21 in K562 cells with RBP2 expression plasmid. (B) qRT-PCR analysis of RBP2 mRNA level after transfection with RBP2 expression plasmid in K562, HL60 and CML-CP and -BP primary cells for 48 h. The primary cells were from the bone marrow of CML-CP and -BP patients. Data are mean ± SEM of 3 independent experiments. (C) qRT-PCR analysis of miR-21 and the loading control U6 snRNA after transfection with RBP2 expression plasmid. Data are mean ± SEM of 3 independent experiments. (D) Predicted binding site of RBP2 in the miR-21 promoter. (E) MiR-21 promoter and miR-21 mutated activity with RBP2 expression plasmid transfection in K562 and HL60 cells. Luciferase activities were determined at 48 h and normalized by Renilla luciferase activity. (F) EMSA assay for the binding of RBP2 to miR-21 promoter. (G) ChIP assay for binding RBP2 to miR-21 promoter in K562 and HL60 cells. (H) The binding of RBP2 and H3K4me3/2 to miR-21 promoter after RBP2 expression plasmid transfection. (I) Western blot analysis of global H3K4me3/2 protein expression in K562 and HL60 cells after transfection with RBP2 expression plasmid. The results are from 3 independent experiments. *P< 0.05, ***P < 0.001.