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Histone demethylase RBP2 decreases miR-21 in blast crisis of chronic myeloid leukemia.

Zhou M, Zeng J, Wang X, Wang X, Huang T, Fu Y, Sun T, Jia J, Chen C - Oncotarget (2015)

Bottom Line: In this study, we found that the histone H3 lysine 4 (H3K4) demethylase RBP2 (also called JARID1A and KDM5A) is underexpressed in CML-BP.This in turn activated PDCD4.In conclusion, RBP2 epigenetically downregulated miR-21 in blast transformation of CML.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Qilu Hospital of Shandong University, Jinan, Shandong, P. R. China.

ABSTRACT
Chronic myeloid leukemia in the blastic phase (CML-BP) responds poorly to clinical treatments and is usually fatal. In this study, we found that the histone H3 lysine 4 (H3K4) demethylase RBP2 (also called JARID1A and KDM5A) is underexpressed in CML-BP. The RBP2 histone demethylase stimulates leukemia cell differentiation and inhibits cell proliferation. We identified miR-21 was directly downregulated by RBP2 and found that miR-21 downregulated PDCD4 expression in leukemia cells. By binding to miR-21 promoter and by demethylating of trimethylated H3K4 at the miR-21 locus, RBP2 downregulated miR-21 expression. This in turn activated PDCD4. In conclusion, RBP2 epigenetically downregulated miR-21 in blast transformation of CML.

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

Mentions: Increasing studies have shown that miRNAs are pivotal in leukemogenesis. To investigate the mechanisms by which RBP2 promotes cell differentiation and inhibits proliferation, we examined the effect of RBP2 overexpression on miRNA expression profiles. We compared miRNA expression levels in RBP2 and control plasmid-treated K562 cells by miRNA microarray analysis and found miRNA genes that were up- or downregulated by RBP2 overexpression. miR-21 was significantly downregulated by RBP2 overexpression (Figure 3A). miR-21 expression was significantly reduced with upregulated RBP2 in K562 and HL60 cells (Figures 3B, C) and in primary cells from the bone marrow of CML-CP and CML-BP patients (Figures 3B, C). Furthermore, with RBP2 overexpression, the level of RBP2 was lower in CML-BP than CML-CP cells (Figure 3B), which suggests that RBP2 expression was decreased during CML progression. Therefore, miR-21 is downregulated by RBP2, so it may be a target gene of RBP2.


Histone demethylase RBP2 decreases miR-21 in blast crisis of chronic myeloid leukemia.

Zhou M, Zeng J, Wang X, Wang X, Huang T, Fu Y, Sun T, Jia J, Chen C - Oncotarget (2015)

MiR-21 is directly and epigenetically downregulated by RBP2(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.
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Figure 3: MiR-21 is directly and epigenetically downregulated by RBP2(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.
Mentions: Increasing studies have shown that miRNAs are pivotal in leukemogenesis. To investigate the mechanisms by which RBP2 promotes cell differentiation and inhibits proliferation, we examined the effect of RBP2 overexpression on miRNA expression profiles. We compared miRNA expression levels in RBP2 and control plasmid-treated K562 cells by miRNA microarray analysis and found miRNA genes that were up- or downregulated by RBP2 overexpression. miR-21 was significantly downregulated by RBP2 overexpression (Figure 3A). miR-21 expression was significantly reduced with upregulated RBP2 in K562 and HL60 cells (Figures 3B, C) and in primary cells from the bone marrow of CML-CP and CML-BP patients (Figures 3B, C). Furthermore, with RBP2 overexpression, the level of RBP2 was lower in CML-BP than CML-CP cells (Figure 3B), which suggests that RBP2 expression was decreased during CML progression. Therefore, miR-21 is downregulated by RBP2, so it may be a target gene of RBP2.

Bottom Line: In this study, we found that the histone H3 lysine 4 (H3K4) demethylase RBP2 (also called JARID1A and KDM5A) is underexpressed in CML-BP.This in turn activated PDCD4.In conclusion, RBP2 epigenetically downregulated miR-21 in blast transformation of CML.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Qilu Hospital of Shandong University, Jinan, Shandong, P. R. China.

ABSTRACT
Chronic myeloid leukemia in the blastic phase (CML-BP) responds poorly to clinical treatments and is usually fatal. In this study, we found that the histone H3 lysine 4 (H3K4) demethylase RBP2 (also called JARID1A and KDM5A) is underexpressed in CML-BP. The RBP2 histone demethylase stimulates leukemia cell differentiation and inhibits cell proliferation. We identified miR-21 was directly downregulated by RBP2 and found that miR-21 downregulated PDCD4 expression in leukemia cells. By binding to miR-21 promoter and by demethylating of trimethylated H3K4 at the miR-21 locus, RBP2 downregulated miR-21 expression. This in turn activated PDCD4. In conclusion, RBP2 epigenetically downregulated miR-21 in blast transformation of CML.

Show MeSH
Related in: MedlinePlus