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, 8 (63), 106527-106537
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Inhibition of EZH2 Triggers the Tumor Suppressive miR-29b Network in Multiple Myeloma

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Inhibition of EZH2 Triggers the Tumor Suppressive miR-29b Network in Multiple Myeloma

Maria Angelica Stamato et al. Oncotarget.

Abstract

Downregulation of tumor suppressor (TS) microRNAs (miRNAs) commonly occurs in human cancer, including multiple myeloma (MM). We previously demonstrated that miR-29b is a relevant TS miRNA, whose expression in MM cells is inhibited by HDAC4-dependent deacetylation. Here, we provide novel insights into epigenetic mechanisms suppressing miR-29b in MM. In MM patient-derived plasma cells, we found inverse correlation between miR-29b and EZH2 mRNA expression. Both siRNAs and pharmacologic inhibitors of EZH2 led to miR-29b upregulation, and this effect was ascribed to reduced H3K27-trimethylation (H3K27me3) of miR-29a/b-1 promoter regions. Induction of miR-29b upon EZH2 inhibition occurred together with downregulation of major miR-29b pro-survival targets, such as SP1, MCL-1 and CDK6. Knock-down of the EZH2-interacting long non-coding RNA MALAT1 also reduced H3K27me3 of miR-29a/b-1 promoter, along with induction of miR-29b and downregulation of miR-29b targets. Importantly, inhibition of miR-29b by antagomiRs dramatically reduced in vitro anti-MM activity of small molecule EZH2-inhibitors, indicating that functional miR-29b is crucial for the activity of these compounds. Altogether, these results disclose novel epigenetic alterations contributing to the suppression of miR-29b molecular network, which can be instrumental for the development of rationally designed miRNA-based anti-MM therapeutics.

Keywords: EZH2; miR-29b; miRNA; microRNA; multiple myeloma.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Inverse correlation between EZH2 and miR-29b in MM patient-derived plasma cells
Correlation of endogenous miR-29b levels with EZH2 (A), EZH1 (B) and MMSET (C) mRNA levels, determined by high density microarray analysis of mRNA or miRNA expression in GSE73454 (for miR-29b) and GSE73452 (for EZH2 mRNA) datasets. Log values of raw data are reported in graph. R = regression coefficient.
Figure 2
Figure 2. Inhibitory effect of EZH2 on miR-29b expression
QRT-PCR analysis of EZH2 (A) and miR-29b (B) expression levels in AMO-BZB and JJN3 cells, 24 hours after transfection with 100 nM scrambled siRNAs (SCR) or EZH2-targeting siRNAs (siEZH2#1 and siEZH2#2). (C) QRT-PCR of miR-29b levels, 24 hours after treatment of JJN3 with 2 μM DZnep, 5 μM GSK343 or 5 μM EPZ005687; WB shows the levels of H3K27me3 and total histone H3 in JJN3-treated cells. (D) WB analysis of SP1, CDK6 and MCL-1, 24 hours after transfection of JJN3 or AMO-BZB cells with 100 nM scrambled siRNAs (SCR) or EZH2-targeting siRNAs (siEZH2#1 and siEZH2#2); GAPDH was used as loading control. (E) WB analysis of SP1, CDK6 and MCL-1, 24 hours after treatment of JJN3 with 2 μM DZnep, 5 μM GSK343 or 5 μM EPZ005687; GAPDH was used as loading control. (F) QRT-PCR analysis of miR-29b expression in AMO-BZB cells, 24 hours after transfection with 2.5 μg of pEZ-M06-EZH2 plasmid (V-EZH2) or of the empty vector (V-CNT). The blot shows levels of EZH2 in AMO-BZB cells, 24 hours after transfection. *P < 0.01.
Figure 3
Figure 3. EZH2 binds miR-29a/b-1 promoter and regulates its H3K27me3
(A) Chip assay using an EZH2 antibody, or IgG isotypic control, was performed in AMO-BZB cells. Results are the average of three independent experiments performed in triplicate and show enrichment of EZH2 at miR-29a/b-1 promoter regions. *P < 0.01. (B) Chip assay using an H3K27me3 antibody or IgG isotypic control, in AMO-BZB cells treated with DMSO or 2 μM DZNep for 24 hours. Results are the average of three independent experiments performed in triplicate and show reduction of H3K27 trimethylation at miR-29a/b-1 promoter regions after inhibition of EZH2 with DZNep. *P < 0.01 respect to DMSO-treated cells.
Figure 4
Figure 4. MALAT1 knock-down upregulates miR-29b expression
(A) QRT-PCR analysis of miR-29b and MALAT1 in AMO-BZB cells after transduction with an empty vector (V-CNT) or a vector containing MALAT1 cDNA (V-MALAT1). QRT-PCR analysis of MALAT1 (B) or miR-29b (C) expression levels, 72 hours after treatment of AMO-BZB cells with 2.5 μM naked MALAT1 ASOs at the indicated concentrations. *P < 0.01. (D) Chip assay using an H3K27me3 antibody or IgG isotypic control, 72 hours after treatment of AMO-BZB cells with naked MALAT1 ASOs. Results are the average of three independent experiments performed in triplicate and show reduction of H3K27 trimethylation at miR-29a/b-1 promoter regions after MALAT1 knock-down with ASOs. *P < 0.01 respect to ASO-control treated cells. (E) WB analysis of SP1, CDK6 and MCL-1, 72 hours after treatment of AMO-BZB cells with naked MALAT1 ASOs or control ASOs. GAPDH was used as loading control.
Figure 5
Figure 5. miR-29b antagonism impairs in vitro anti-MM activity of EZH2 inhibitors
(A) QRT-PCR analysis of miR-29b expression levels in AMO-BZB transduced with GFP control or miR-29b inhibitors (antimiR-29b). Trypan blue exclusion assay (B) and CTG viability assay (C) were performed in AMO-BZB cells stably transduced with GFP control or miR-29b inhibitors (anti-miR-29b), 72 hours after treatment with 2 μM DZnep, 5 μM GSK343 or 5 μM EPZ005687. *P < 0.05.
Figure 6
Figure 6. Graphic overview of the inhibitory effect played by EZH2 and MALAT1 on miR-29b expression
The cartoon shows that MALAT1 and EZH2 negatively regulates miR-29b expression by increasing H3K27me3 of miR-29a/b-1 promoter, thus affecting levels of miR-29b oncogenic targets SP1, MCL-1 and CDK6.

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