TEL-AML1 regulation of survivin and apoptosis via miRNA-494 and miRNA-320a

Abstract

There is increasing evidence that miRNA and transcription factors interact in an instructive fashion in normal and malignant hematopoiesis. We explored the impact of TEL-AML1 (ETV6-RUNX1), the most common fusion protein in childhood leukemia, on miRNA expression and the leukemic phenotype. Using RNA interference, miRNA expression arrays, and quantitative polymerase chain reaction, we identified miRNA-494 and miRNA-320a to be up-regulated upon TEL-AML1 silencing independently of TEL expression. Chromatin immunoprecipitation analysis identified miRNA-494 as a direct miRNA target of the fusion protein TEL-AML1. Using bioinformatic analysis as well as functional luciferase experiments, we demonstrate that survivin is a target of the 2 miRNAs. miRNA-494 and miRNA-320a were introduced to the cells by transfection and survivin expression determined by Western blot analysis. These miRNAs blocked survivin expression and resulted in apoptosis in a similar manner as TEL-AML1 silencing by itself; this silencing was also shown to be Dicer-dependent. miRNAs-494 and -320a are expressed at lower levels in TEL-AML1+ leukemias compared with immunophenotype-matched nonTEL-AML1 acute lymphoblastic leukemia subtypes, and within TEL-AML1+ leukemias their expression is correlated to survivin levels. In summary our data suggest that TEL-AML1 might exert its antiapoptotic action at least in part by suppressing miRNA-494 and miRNA-320a, lowering their expression causing enhanced survivin expression.

Figure 1

siRNA-mediated silencing of TEL-AML1 affects miRNA expression. (A) Schematic representation of the siRNA sequence used to target TEL-AML1 and the localization within the TEL-AML1 translocation breakpoint mRNA (position at bp 15-33, accession no. S78496). A siRNA with no human targets was used as control (see Diakos et al.). (B) Depletion of TEL-AML1 using siRNA. Western analysis was performed from REH cell lysates treated with the control siRNA (left lane) or functional siRNA targeting TEL-AML1 (right lane). TEL-AML1 protein was detected using an anti-TEL antibody from ATLAS and an anti-tubulin antibody for loading control. (C) TEL-AML1 affects miRNA expression. The expression profile of REH cell was analyzed after TEL-AML1 depletion by 2 color miRNA arrays. The fold changes of the 4 most up-regulated as well as those of the 4 most down-regulated miRNAs are shown (mean of 2 experiments). (D) miRNA RT-PCR (Applied Biosystems) was performed to verify the changes in miRNA-494 and miRNA-320a expression upon TEL-AML1 silencing. The data shown depicts the average fold change from 3 independent experiments, using U6 snRNA as a calibration control.

Figure 2

TEL-AML1 directly targets miRNA promoters. (A) ChIP-chip analysis was performed using the TEL-AML1 expressing cell line REH using an anti-TEL antibody. This cell line has a deletion in the other TEL allele, so only TEL-AML1 targets are pulled-down. Two independent experiments were performed, and anti-TEL-AML1 pull-downs and input DNA controls were cohybridized to the ncRNA tiling arrays. Following normalization and smoothing, the log2-ratios of TEL-AML1 pull-downs versus controls from experiment 1 were graphed against those from experiment 2 on an x/y graph, with density shading. A Pearson correlation coefficient of 0.27 (P < 10⁻⁷) between the 2 replicate experiments was observed as shown in this plot, also exemplified by a clustering of data along a slope of x = y (B) Scrutiny of the pull-down peaks revealed TEL-AML1 binding to miRNA regions, the strongest pull-down was detected for the miRNA-139 promoter. Lower but significant signals were found for 99 other miRNAs, shown here is the peak in a 5′ region of miRNA-494. (C) TEL-AML1 binds the promoters of miRNAs. The average log ratio of the binding intensity from 3 experiments was calculated and the 8 miRNAs promoters with the highest affinity to bind TEL-AML1 are shown, among these is miRNA-494. Standard errors are calculated from 3 separate experiments. (D) miRNA-494 and miRNA-320a are direct and functional targets of TEL-AML1. miRNA promoters identified to bind TEL-AML1 (direct targets, y axis; only those targets significantly enriched in ChIP pull downs are shown) were plotted against miRNA expression changes upon TEL-AML1 silencing. Log₂ ratios are shown. (E) Using conventional PCR (supplemental Table 1) we analyzed TEL-AML1 pull-down and input from ChIP experiments. We designed primers to test the miRNA-494 and miRNA-320a that were shown to be direct targets of TEL-AML1 in the ChIP experiments. Panel E shows the fold increase of TEL-AML1 binding to the promoters of miRNA-494 and miRNA-320a as it was compared with the input (in triplicate with SE bars), thereby confirming these miRNAs as direct targets of TEL-AML1. The combined data from these experimental approaches distinguishes miRNA-494 and miRNA-320a both as direct and functional targets. miRNA-320a is indicated as miRNA-320 on the figures, as it is termed in the Sanger database.

Figure 3

miRNA-494 and miRNA-320a affect survivin expression and apoptosis. (A) Bioinformatic target prediction analysis identifies survivin as a target of miR-494 and miRNA-320a; the survivin 3′ UTR complementary to the miRNA sequence is shown (using Targetscan). (B) Both miRNA-494 and miRNA-320a can regulate survivin expression. miRNA mimics for miRNA-494 and miRNA-320a were transfected into the REH cells and survivin expression was analyzed by Western blot analysis using a rabbit anti-survivin antibody (Cell Signaling Technology). The miRNA with no human targets were used as control. For loading controls, an equal number of cells from each experimental sample were lysed, and the same volume of the same lysate was applied to a separate gel to probe for tubulin. (C) miRNA-494 and miRNA-320a induce apoptosis. miRNA mimics for miRNA-494 and miRNA-320a were transfected into the REH cells and apoptosis was assessed by FACS analysis of annexin V–FITC/PI staining of control-treated (miRNA with no human targets, see “Methods”), and mimics for miRNA-494 and miRNA-320a. Data are shown from 1 of 3 representative experiments and indicate early apoptosis (annexin V single-positive cells). Percentages of annexin V–positive cells are indicated in the figure (SEs are under 2%). Percentages refer to the cells within the top left and bottom left quadrants, and top right and bottom right, respectively. (D) TEL-AML1 regulation of survivin is Dicer1 dependent. siRNA silencing of Dicer1 restores survivin expression in TEL-AML1 depleted cells indicating the miRNA dependence of this effect. For loading controls, an equal number of cells from each experimental sample were lysed, and the same volume of the same lysate was applied to a separate gel to probe for tubulin. (E) Luciferase vectors containing portions of the survivin (BIRC5) 3-UTR was used to demonstrate that survivin is a direct target of miRNA-494 and miRNA-320a. Both miRNAs were able to block luciferase expression significantly when they were cotransfected into REH cell together with the BIRC5 3-UTR luciferase-expressing vector (WT BIRC5). A mutated sequence and the empty vector were used as controls (MUT BIRC5 and pMir Reporter, respectively), and both had equivalent levels of luciferase activity. The Y-axis displays relative luminescence units, which are normalized per unit of beta-galactosidase activity. SEs from 3 separate experiments are shown. The wild-type target sequence had at minimum average 6-fold lower luminescence compared with the mutant targets, and miRNA targeted to the mutant sequence did not significantly impact luciferase activity compared with the empty vector control. Sequences used to create the plasmids are shown in supplemental Table 1. The mismatch mutant vectors contain 2 mismatch bases from the miRNA seed sequence.

Figure 4

The impact of TEL-AML1 silencing upon miRNA-494 and miRNA-320a is independent of TEL expression. (A) HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) with TEL or TEL-AML1 plasmids cloned into pcDNA 3.1 according to manufacturer's protocol exactly as explained in Diakos et al. Cells were lysed in radio-immunoprecipitation assay buffer containing proteinase inhibitors (Complete, Roche) with subsequent western analysis. Transient expression of proteins was confirmed by using TEL antibody (AB23465-100 Abcam) antibody and the LI-COR detection system (LI-COR Odyssey). (B) An ETV6 (TEL) expressing vector was introduced into REH cells by lipofection (lane 1) and the TEL-AML1 expression was silenced using siRNA (lane 2). The expression of TEL and TEL-AML1 was analyzed by Western blot analysis using an antibody against TEL. An anti-tubulin antibody was used for loading control on the same gel (using the LI-COR system). (C-D) The expression of miRNA-320 (C) and miRNA-494 (D) were analyzed by miRNA Taqman PCR. The black bars represent the results from lanes 1 to 3 for the HEK293T experiment shown in Figure 5A, and the gray bars represent the result from lanes 1 and 2 for the REH cell experiment in Figure 5B. miRNA levels are displayed relative to the empty vector HEK293a result.

Figure 5

miR-494 and miR-320a expression in leukemia samples, and a model of action. (A) RNA was isolated from diagnostic leukemia samples from 11 TEL-AML1 positive and 15 TEL-AML1 negative cALL patients, and miRNA expression for miR-494 and miR-320a were analyzed by quantitative PCR. Shown are box-and-whisker plots of log₁₀-transformed data; the box contains 50% of the data separated by the median, and the whiskers contain the remaining 25% at each side. TEL-AML1 leukemias expressed less miRNA-494 (P = .04) and miRNA-320a (P = .03) than other cALLs. (B) Scatterplot of the relationship of miRNA-320a (measured by Taqman) and survivin protein (measured by ELISA) among 28 cryopreserved TEL-AML1 leukemia cell samples. Two samples that were outliers were removed to “center” the remainder of the data. miRNA-320a and survivin were correlated (Spearman corrrelation: −0.38, P = .015). (C) Schematic presentation of the TEL-AML1, miRNA-494, and miRNA-320a impact upon survivin expression and cell survival. TEL-AML1 binds miRNA-494 and miRNA-320a promoters and exerts its transcriptional suppressor activity. These block miRNA-494 and miRNA-320a expression and release miRNA control of survivin expression resulting in increased resistance from apoptosis.