MiR-27a Functions as a Tumor Suppressor in Acute Leukemia by Regulating 14-3-3θ

<div><p>MicroRNAs (miRs) play major roles in normal hematopoietic differentiation and hematopoietic malignancies. In this work, we report that miR-27a, and its coordinately expressed cluster (miR-23a∼miR-27a∼miR-24-2), was down-regulated in acute leukemia cell lines and primary samples compared to hematopoietic stem-progenitor cells (HSPCs). Decreased miR-23a cluster expression in some acute leukemia cell lines was mediated by c-MYC. Replacement of miR-27a in acute leukemia cell lines inhibited cell growth due, at least in part, to increased cellular apoptosis. We identified a member of the anti-apoptotic 14-3-3 family of proteins, which support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad, as a target of miR-27a. Specifically, miR-27a regulated 14-3-3θ at both the mRNA and protein levels. These data indicate that miR-27a contributes a tumor suppressor-like activity in acute leukemia cells via regulation of apoptosis, and that miR-27a and 14-3-3θ may be potential therapeutic targets.</p> </div

The miR-23a cluster does not regulate 14-3-3ζ, or other 14-3-3 isotypes.

<p>(A) and (B) The effect of the miR-23a cluster on (A) 14-3-3ζ and (B) 14-3-3γ protein expression was measured via Western blot. The top panels are representative blots from one experiment, and the bottom panels are graphs of normalized protein expression from 3 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (C) The schematics show predicted miR-27a and miR-24 binding sites in the CDS and 3′UTR of 14-3-3ζ (top panel). HEK293T cells were co-transfected with 14-3-3ζL-157, 14-3-3ζL-627, or 14-3-3ζL-1075 (500 ng, white bars) alone and with 25 nM of miR-23a (light grey bars) or miR-24 (dark grey bars); HEK293 cells were also co-transfected with 14-3-3ζL-1266 (500 ng, white bars) alone and with 25 nM of miR-23a (light grey bars) or miR-27a (black bars) (bottom panel). Normalized mean Luc expression (±SEM, n≥3) is represented on the graphs.</p

MiR-27a regulates14-3-3θ via a single binding site in the 3′UTR.

<p>(A)–(C) Expression of mature miR-27a and 14-3-3θ mRNA were measured via microarray. Correlation plots of 14-3-3θ and miR-27a expression in (A) pre-B-ALL (n = 37), (B) T-ALL (n = 26), and (C) AML (n = 39) cell lines and primary samples are shown. Significant inverse correlation was determined via Pearson r (r<<b>−</b>0.30, p<0.05*). (D) The effect of miR-27a on 14-3-3θ protein expression was measured via Western blot; the top panel is a representative experiment, and the bottom panel is a graph of normalized protein expression from 3 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (E) A schematic showing the predicted miR-27a binding site in the 3′UTR of 14-3-3θ (bottom). HEK293T cells were co-transfected with 14-3-3θL-27a or 14-3-3θL-27a-del (500 ng, white bars) alone and with 25 nM of miR-23a (grey bars) or miR-27a (black bars) (top). Normalized mean Luc expression (±SEM) is represented on the graphs. (F) The 14-3-3θ ORF rescues miR-27a-induced cell death. FUGW and FUGW/miR-27a transduced K562 clones were transfected with control plasmid (black bars) or 2 µg 14-3-3θ ORF (white bars), and stained with PI. FUGW/miR-27a transduced clones expressing the 14-3-3θ ORF had significantly less PI+ cells compared to those clones transfected with control vector. Significant differences were determined by Student's t-test (n≥3, p<0.05*).</p

Effects of c-MYC on expression of the miR-23a cluster.

<p>(A) Knock down of c-MYC expression in P493B cells by doxycycline-treatment. Cells were treated with 0 µg/ml (white bar), 0.1 µg/ml (grey bar) or 0.3 µg/ml (black bar) doxycycline for 48 hr, harvested, and total RNA isolated. Levels of miR-23a cluster member expression were measured via qRT-PCR, using U18 as an endogenous control, and normalized to untreated (0 µg/ml Dox) cells (2^−ΔΔCt = 1). (B) and (C) The effects of pharmacologic inhibition of c-MYC on miR-23a (B) and miR-27a (C) in REH, KOPN8, SUPB15, and K562 cell lines were determined by treatment with 2 doses of 10058-F4 (25 µM [grey bar] and 50 µM [black bar]) or vehicle (white bar) for 48 hr. Fold-expression of mature miR-23a and miR-27a were assessed by qRT-PCR and analyzed as above. Effect of the c-MYC inhibitor on miR-23a and miR-27a expression was determined by comparison of treated cells to the mean expression (±SEM) of vehicle-treated cells Significance was determined by a Student's t-test where p<0.05 (*) indicated significance (n = 2–6 independent experiments). (D) and (E) The effect of siRNA knock-down of endogenous c-MYC on expression of miR-23a (D) and miR-27a (E) in K562, Karpas45, and Molt16 cells were assessed by transfection with 50 nM (grey bar) or 100 nM (black bar) of an siRNA-c-MYC pool or 100 nM of control siRNA (white bar). Expression levels of miR-23a and miR-27a and significance of results were assessed as in (B) and (C). n≥3 independent experiments for all samples.</p

Enforced expression of miR-27a in pre-B-ALLs and T-ALLs.

<p>(A) Viability of FUGW and FUGW/miR-27a transduced REH (pre-B-ALL) clonal populations was measured at indicated times as above. Statistical differences in viability over time in FUGW and FUGW/miR-27a K562 colonies were determined by a 1-way ANOVA test (p<0.0001*). (B) REH cells were stained with AnnexinV and 7AAD and FACS-analyzed. Each data point is a replicate for the designated clonal population. Significant differences between FUGW and FUGW/miR-27a clones were compared as above. (C) FUGW and FUGW/miR-27a transduced Molt16 (T-ALL) cells were FACS-analyzed for GFP expression and PI staining. GFP expression positively correlated with PI+ cells in FUGW/miR-27a transduced cells (Pearson r = 0.9965, p<0.0001) compared to FUGW control cells which did not demonstrate this correlation. (D) GFP expression was monitored via FACS over time in FUGW and FUGW/miR-27a Molt16 GFP−/GFP+ mixed populations.</p

Enforced expression of miR-27a in AMLs.

<p>(A) Effects on proliferation were demonstrated by plating 10⁵ cells/ml in suspension cultures (Day 0) and counting viable cells (via trypan blue exclusion) on Days 1, 2, and 4 (n = 3 per colony). Growth was plotted as the mean viable cell number ±SD. Colony #'s were arbitrary. (B) MiR-27a expression levels in vehicle- and Dox-treated (0.30 µg/ml) TG-27a transduced K562 cells was determined by qRT-PCR and are represented as mean fold expression (2^−ΔΔCt) ±SEM. (C) Untransduced K562 cells (WT), K562 cells transduced with a control tet-inducible plasmid, and K562 cells transduced with the TG-27a construct were treated with increasing doses of Dox (as indicated). Cell growth of control- or tet-inducible construct-transduced cells was normalized to that of untransduced K562 cells. (D) Viability of FUGW and FUGW/miR-27a transduced stable K562 clones was measured at indicated times after isolation from methylcellulose colonies. Each data point represents 1 clonal population (i.e. cells grown up from 1 colony and followed over time). Statistical differences in viability over time in FUGW and FUGW/miR-27a K562 colonies were determined by a 1-way ANOVA test (p<0.0001*). (E) FUGW and FUGW/miR-27a K562 clonal populations were stained with PI and FACS-analyzed. Each data point on the graph represents percent PI of an individual clonal population. The mean (±SEM) of FUGW clones (n = 4) was compared to FUGW/miR-27a clones (n = 7), and significant differences were assessed via student's t-test (p<0.05*). (F) K562, TF1, and HL60 AML cells were stained with AnnexinV and 7AAD and FACS-analyzed. Each data point represents the % AnnexinV⁺/7AAD⁻ population within an individual clone. Significant differences between FUGW and FUGW/miR-27a clones were compared as above.</p

MiR-23a cluster expression in pre-B-ALL, T-ALL, and AML cell lines and primary samples.

<p>Expression of miR-27a (A), miR-23a (B), and miR-24 (C) was determined via microarray analysis in pre-B-ALL, T-ALL, and AML. (A) Average miR-27a expression (±SEM, p<0.05*) was compared to expression in normal CD34+ HSPCs (5.76±0.35, represented by black line) in pre-B-ALL cell lines (n = 36 [replicates of 9 cell lines]), pre-B-ALL primary samples (n = 16), T-ALL cell lines (n = 23 [replicates of 5 cell lines]), T-ALL primary samples (n = 13), AML cell lines (n = 24 [replicates of 7 cell lines]), and AML primary samples (n = 13). (B) Average miR-23a expression (±SEM, p<0.05*) was compared to normal CD34+ HSPCs (9.76±0.19, represented by black line). (C) Average miR-24 expression (±SEM, p<0.05*) was compared to expression in normal CD34+ HSPCs (10.23±0.14). n values for (B) and (C) were as in (A).</p

Expression levels of mature miR-27a in AML, pre-B-ALL, and T-ALL cell lines and primary samples.

<p>Total RNA was isolated from human AML (A), pre-B-ALL (B), and T-ALL (C) cell lines (black bars) and primary cases (primary patient cases identified with unique numbers, white bars). MiR-27a expression levels were normalized to mean levels in normal human CD34+ HSPCs (2^−ΔΔCt = 0.9756±0.022, black line). A Student's t-test was used to determine the significance of the difference in mean miR-27a expression (±SEM) between each leukemia samples and normal CD34+ HSPCs; significance is indicated as p<0.05 (*). n≥3 independent experiments in all cases except for the 10 primary AMLs which were analyzed in triplicate due to limited sample quantity.</p

MiR-24, but not miR-27a regulates 14-3-3β via sites in the 3′UTR and CDS.

<p>(A)–(C) Expression of mature miR-24 and 14-3-3β mRNA were measured via microarray. Correlation plots of 14-3-3β and miR-24 expression in (A) pre-B-ALL (n = 37), (B) T-ALL (n = 20), and (C) AML (n = 36) cell lines and primary samples are shown. Significant inverse correlation was determined via Pearson r (r<−0.03, p<0.05*). (D) The effect of miR-23a cluster members on endogenous 14-3-3β protein expression was measured via Western blot. The left panel is a representative blot from one experiment, and the right panel is a graph of normalized protein expression from 4 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (E) A schematic showing predicted miR-27a binding sites in the 3′UTR of 14-3-3β (top). HEK293T cells were co-transfected with 14-3-3βL-1537 or 14-3-3βL-1537-del (middle panel), and 14-3-3βL-2061 or 14-3-3βL-2061-del (bottom panel) (500 ng, white bars) alone and with 25 nM of miR-23a (grey bars) or miR-27a (black bars). Normalized mean Luc expression (±SEM, n≥3) is represented on the graph. (F) A schematic shows predicted miR-24 binding sites in the CDS and 3′UTR of 14-3-3β (top). HEK293T cells were co-transfected with 14-3-3βL-3′UTR (or deletion mutants 1 and 2; middle panel) or 14-3-3βL-CDS (or deletion mutants 1 and 2; bottom panel), (500 ng, white bars) alone and with 25 nM of miR-24 (grey bars) or miR-23a (black bars). Normalized mean Luc expression (±SEM, n≥3) is represented.</p