ATM is down-regulated by N-Myc–regulated microRNA-421

Abstract

Ataxia-telangiectasia mutated (ATM) is a high molecular weight protein serine/threonine kinase that plays a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks. Little is known about the regulatory mechanisms for ATM expression itself. MicroRNAs are naturally existing regulators that modulate gene expression in a sequence-specific manner. Here, we show that a human microRNA, miR-421, suppresses ATM expression by targeting the 3′-untranslated region (3′UTR) of ATM transcripts. Ectopic expression of miR-421 resulted in S-phase cell cycle checkpoint changes and an increased sensitivity to ionizing radiation, creating a cellular phenotype similar to that of cells derived from ataxia-telangiectasia (A-T) patients. Blocking the interaction between miR-421 and ATM 3′UTR with an antisense morpholino oligonucleotide rescued the defective phenotype caused by miR-421 overexpression, indicating that ATM mediates the effect of miR-421 on cell cycle checkpoint and radiosensitivity. Overexpression of the N-Myc transcription factor, an oncogene frequently amplified in neuroblastoma, induced miR-421 expression, which, in turn, down-regulated ATM expression, establishing a linear signaling pathway that may contribute to N-Myc-induced tumorigenesis in neuroblastoma. Taken together, our findings implicate a previously undescribed regulatory mechanism for ATM expression and ATM-dependent DNA damage response and provide several potential targets for treating neuroblastoma and perhaps A-T.

Ataxia-telangiectasia mutated (ATM) kinase plays a hierarchical regulatory role in the double-strand break (DSB)-induced DNA damage response in which ATM transduces a DSB damage/repair signal to downstream effector machinery by phosphorylating critical protein substrates (1 –4). ATM mutations, which usually result in loss of ATM protein expression (5), lead to the autosomal recessive progressive neurodegenerative disease ataxia-telangiectasia (A-T) (6, 7). Both homozygotes and heterozygotes are at an increased risk for cancer (8). ATM has been reported to be regulated by a transcription factor, E2F-1, (9) and the ATM gene is also reported to be subject to epigenetic silencing such as by methylation of the ATM promoter (10, 11), suggesting that ATM can also be up-regulated at the transcriptional level under some circumstances. MicroRNAs regulate gene expression through inhibition of translation or degradation of the targeted mRNA (12, 13). Physiological functions of microRNAs have been observed in normal and lineage-targeted development (14) as well as in the context of human cancers (15). In this study, we demonstrate that miR-421 targets the 3′-untranslated region (3′UTR) of ATM and down-regulates its expression, whereas miR-421 expression is driven by the N-Myc transcription factor, an oncogene that is frequently amplified in neuroblastoma cells.

Results

MiR-421 Suppresses ATM Expression by Targeting 3′UTR of ATM.

To explore the possibility that microRNAs might regulate ATM expression, we searched the 3′UTR of the human ATM gene for microRNA-binding motifs using the MicroCosm Targets program (EMBL-EBI). Nine nucleotides at the 5′-end of hsa-miR-421 (miR-421) were perfectly complementary to the target sequence in the 3′UTR of ATM (including the “seed sequence” from positions 2–8) (Fig. 1A). This suggested that ATM might be a target for miR-421. To validate this in silico prediction, we cloned the ATM 3′UTR portion containing the miR-421 target site into a Renilla luciferase reporter construct (Fig. 1B) and established a luciferase reporter assay following cotransfection of reporter constructs with precursor miR-421 (pre-miR-421) into HeLa cells. A significant reduction (30%) in the luciferase activity of the reporter construct containing the ATM 3′UTR was observed in the presence of miR-421, whereas no changes were noted in the luciferase activity of the unmodified construct (pRL) with miR-421 expression (Fig. 1C). Deletion of six nucleotides of seed sequence (Δ6) led to the loss of reduction in miR-421-mediated luciferase activity (Fig. 1C). To further confirm that ATM is a target for miR-421, we examined the endogenous ATM protein level by immunoblot after transiently transfecting pre-miR-421 into HeLa cells. As shown in Fig. 1D, the ATM expression level decreased as the concentration of transfected pre-miR-421 was increased. As an indication of ATM kinase activity (16), phosphorylation of SMC1 at the serine-966 residue (pS966-SMC1) was measured following DNA damage by 10-Gy irradiation (IR). A significant reduction in the pS966-SMC1 was observed when pre-miR-421 was introduced into HeLa cells followed by IR, as compared with the introduction of a nonrelevant control pre-miR precursor (Fig. 1E). ATM mRNA levels were measured by quantitative real-time PCR and were not decreased in the presence of miR-421 (Fig. 1F), suggesting that miR-421 down-regulates ATM at a translational rather than transcriptional level.

Fig. 1.

miR-421 suppresses ATM expression by targeting ATM 3′UTR. (A) Mature miR-421 sequences and recognition sites within 3′UTR of ATM. The seed sequence of miR-421 is shown in the box. WT and del6 (Δ6) ATM-3′UTR targets are also shown. (B) Constructs of Renilla luciferase [Luc; unmodified construct (pRL)-CMV] containing WT or 6-nt deleted (Δ6) ATM 3′UTR. (C) Luciferase (Luc.) activity of pRL and modified constructs containing WT or mutant (Δ6) 3′UTR. Luciferase constructs were cotransfected with pre-miR-CTL (control, 50 nM) or pre-miR-421 (50 nM) into HeLa cells. Renilla luciferase activity was measured 36 h after incubation and normalized to firefly luciferase. Asterisk indicates significant down-regulation of pre-miR-421 against construct containing WT ATM 3′UTR. (D) Immunoblot of endogenous ATM expression in HeLa cells 96 h after transfection of increasing amounts of pre-miR-421 (using pre-miR-CTL to compensate for equal amounts of total miRs). SMC1 served as a loading control for the blot. (E) Immunoblot of pS966-SMC1 in HeLa cells that were transiently transfected with pre-miR-CTL (100 nM) or pre-miR-421 (100 nM) after 10-Gy IR to activate the DNA damage response. A WT lymphoblastoid cell line (LCL) served as a positive control for SMC1 and ATM protein. (F) Real-time PCR of ATM mRNA from HeLa cells transfected with pre-miR-CTL (100 nM) or pre-miR-421 (100n M). Data were normalized to the level of GAPDH mRNA, and the ratio of ATM/GAPDH in HeLa control cells was set to 1.

MiR-421 Regulates Cell Cycle S-Phase Checkpoint and Cellular Radiosensitivity.

To determine the cellular functions of miR-421, we created an miR421-overexpressing HeLa stable cell line by infecting the cells with an miR421-containing lentivirus and selecting a stable infectant with blasticidin (HeLa/miR-421) (Fig. 2A). We also created a control stable infectant cell line with scrambled shRNA (HeLa/scram) (17). Real-time PCR detected an ∼120-fold increase in the expression of mature miR-421 in the HeLa/miR-421 cells compared with the HeLa/scram control cells (Fig. 2B). Both ATM protein expression and ATM kinase activity, as indicated by the level of post-IR pS966-SMC1, were significantly reduced in the HeLa/miR-421 cells (Fig. 2C).

Fig. 2.

miR-421 regulates cell cycle S-phase checkpoint and cellular radiosensitivity. (A) Scheme of a U6 promoter-driven miR-421 cloned into a lentiviral vector with two LTRs and a selection marker for blasticidin driven by SV40 promoter. (B) Real-time PCR of miR-421 expression in HeLa cells stably overexpressing scrambled shRNA (HeLa/scram) or miR-421(HeLa/miR-421). Data were normalized to an internal control RNU66, and the ratio of miR-421/RNU66 in HeLa/scram cells was set to 1. (C) Immunoblot of ATM and pSMC1 in HeLa/scram and HeLa/miR-421 cells with or without 10-Gy IR. Note the reduction of both ATM and pSMC1 in miR421-overexpressing cells. (D) Analysis of IR-induced cell cycle S-phase checkpoint by FC. Stably overexpressing HeLa/scram and HeLa/miR-421 cells were treated with or without 10 Gy. DNA synthesis at S-phase was labeled with BrdU. (Left Upper and Lower) Results of one experiment representative of three independent experiments. Box R5 indicates the percentage of BrdU⁺ S-phase cells pre- or post-IR. (Right) Summary of change of BrdU⁺ cells pre- and post-IR for HeLa/scram and HeLa/miR-421 cells from three independent experiments, using the algorithm (R5^+IR−R5^−IR)/R5^−IR × 100%. (E) HeLa/scram and HeLa/miR-421 cells were irradiated at the indicated doses, and colony survival was measured after 2 weeks. (F) Effect of miR-421 on proliferation of HeLa cells, as measured by cell population doublings with culture time. (G) Effect of miR-421 on IR-induced cell death, as measured by propidium iodide staining FC. The percentage of propidium iodide-positive cells was normalized to the unirradiated cells in each group.

ATM regulates DNA damage-induced cell cycle checkpoints at G1-S and intra-S phase (18, 19). A hallmark of A-T cells is the failure to arrest DNA synthesis in S-phase following DNA damage and continuous incorporation of nucleotides into DNA despite damage (i.e., radioresistant DNA synthesis) (20, 21). Thus, we anticipated that miR-421 overexpression might regulate DNA damage-induced cell cycle S-phase checkpoints. To assess this, HeLa/scram and HeLa/miR-421 cells were irradiated (10 Gy) to introduce DNA damage and BrdU was used to follow DNA incorporation. As expected, a reduction in the percentage of BrdU-positive cells in S-phase was observed for the HeLa/scram control cells (14.88% pre-IR vs. 12.56% post-IR), indicating a normal block in the DNA synthesis (Fig. 2D, Upper Left and Right); in contrast, an increase in the percentage of BrdU-positive cells in S-phase was observed for HeLa/miR-421 cells (11.67% pre-IR vs. 15.38% post-IR) (Fig. 2D, Lower Left and Right), indicating that miR-421 overexpression overcomes the IR-induced DNA synthesis block and mimics the radioresistant DNA synthesis of A-T cells. The miR421-induced continuous DNA synthesis was also seen with lower doses of IR at 2 and 5 Gy (Fig. S1A). We noticed that the pre-IR percentage of HeLa/miR-421 cells in S-phase was lower than that of control HeLa/scram cells, suggesting that miR-421 might regulate this cell cycle checkpoint independent of DNA damage. Similar results were observed using a human breast cancer cell line, MDA-MB-231, when miR-421 was overexpressed (Fig. S2 A and B).

A clonogenic assay was used to determine whether overexpression of miR-421 affects cellular radiosensitivity. As shown in Fig. 2E, the survival fractions of HeLa/miR-421 cells post-IR (1 and 2 Gy) were significantly reduced relative to those of HeLa/scram control cells. MiR-421 overexpression did not alter the proliferation rate of HeLa cells (Fig. 2F) but increased post-IR cell death (Fig. 2G), which is consistent with the decreased survival fraction in the clonogenic assay. A similar effect of miR-421 on radiosensitivity was observed with MDA-MB-231 cells (Fig. S2C).

Effects of miR-421 on S-Phase Checkpoint and Radiosensitivity Are ATM Dependent.

A single microRNA is predicted to modulate >200 targets of protein expression (22). To further determine whether the effects of miR-421 on cell cycle checkpoints and radiosensitivity are mediated through ATM, we used an antisense morpholino oligonucleotide (AMO) to block the recognition sequence of ATM 3′UTR (Fig. 3A). Treatment of HeLa/miR-421 cells with ATM 3′UTR target site-specific AMO (AMO-ATM) resulted in the abrogation of miR421-mediated down-regulation of ATM expression, as shown by both Western blot and ELISA (Fig. 3B). This effect was not observed when cells were treated with scrambled control AMO (AMO-scram) (Fig. S1B). Following IR, AMO-ATM treatment also resulted in an increase of pS966-SMC1 in HeLa/miR-421 cells (Fig. 3C). Blocking with AMO-ATM further restored the S-phase cell cycle checkpoint and radiosensitivity of the HeLa/miR-421 cells, as shown by radioresistant DNA synthesis assay and clonogenic survival assay (Fig. 3 D and E). Taken together, these AMO-ATM experiments suggest that the effect of miR-421 on the cell cycle S-phase checkpoint and radiosensitivity is mediated through ATM.

Fig. 3.

ATM mediates the effect of miR-421 on cell cycle S-phase checkpoint and radiosensitivity. (A) Schematic working model of ATM 3′UTR that was targeted by an antisense AMO. AMO-ATM was designed to match the miR-421 recognition site of ATM 3′UTR and specifically block the down-regulation of ATM by impeding the binding of mature miR-421. (B) (Left) Immunoblot of ATM expression in HeLa/scram and HeLa/miR-421 cells treated with or without AMO-ATM (2 μM) for 5 days. The fold change in ATM expression is shown below the immunoblot. (Right) ELISA was also used to determine ATM concentration. (C) Immunoblot of pSMC1 in HeLa/scram and HeLa/miR-421 cells treated with AMO-scram (2 μM) or AMO-ATM (2 μM) for 5 days, followed by 10-Gy IR. The fold change in pSMC1 level is shown below the immunoblot. Note the increase of pSMC1 in HeLa/miR-421 cells treated with AMO-ATM. (D) Analysis of cell cycle S-phase checkpoint after treatment of AMO. HeLa/scram and HeLa/miR-421 cells were treated with AMO-scram (2 μM) or AMO-ATM (2 μM) for 5 days and irradiated with increasing doses of radiation (2, 5, and 10 Gy). DNA synthesis was monitored by BrdU incorporation and analyzed by FC. The percentage of BrdU⁺ S-phase cells at the start point (unirradiated) was arbitrarily set to 50%, and all other data were normalized to this point. This plot is representative of three independent experiments. The arrow indicates that AMO-ATM treatment rescues the defect of HeLa/miR-421 cells. (E) Colony survival fraction with exposure to AMO. HeLa/scram and HeLa/miR-421 cells were treated with AMO-scram (2 μM) or AMO-ATM (2 μM) for 5 days, and 500 cells were plated in triplicate; cells were irradiated with increasing doses of radiation, and surviving colonies were scored after 2 weeks. The survival fraction at each radiation dose was normalized to that of the nonirradiated control. The arrow indicates that the AMO rescued the radiosensitivity of HeLa/miR-421 cells.

Transcription Factor N-Myc Up-Regulates miR-421 Expression.

Human miR-421 is located intergenically at chromosome Xq13. Interestingly, another microRNA, miR-374b, is located just 85 bp proximal to miR-421, forming a microRNA cluster that is driven by a single promoter (Fig. 4A). The function of miR-374b is still unknown. To determine which transcription factors might influence miR-421 expression, we performed in silico analysis of the promoter region (including 2 kb upstream of the miR-374b stem loop) using the transcription factor binding site program CONSITE (Materials and Methods). This identified a binding site for N-Myc (an E-box) at −85 nucleotides relative to the miR-374b stem loop (Fig. 4A). To validate this prediction, we cloned a 1-kb DNA fragment of the promoter region into a firefly luciferase reporter construct and examined the effect of N-Myc on miR-421 promoter-driven luciferase activity. Overexpression of N-Myc in HeLa cells activated miR-421 promoter-driven luciferase activity 24 and 48 h after transfection (Fig. 4B). Consistent with the luciferase assay, the expression level of endogenous mature miR-421 was also increased by overexpression of N-Myc in the HeLa cells, as measured by microRNA real-time PCR (Fig. 4C). Most interestingly, ATM protein expression was reduced in HeLa cells that were transiently transfected with N-Myc, as detected by immunoblotting and ELISA (Fig. 4 D and E), strongly suggesting that N-Myc stimulates miR-421 expression, which, in turn, down-regulates ATM expression. Anti-miR-421 inhibitor is a chemically modified antisense oligonucleotide designed specifically to bind to and inhibit endogenous miR-421 molecule. Cotransfection of anti-miR-421 inhibitor into HeLa cells along with N-Myc construct restored the ATM expression that was suppressed in N-Myc-transfected cells, further confirming that miR-421 mediates N-Myc-induced ATM down-regulation (Fig. 4 F and G). We also noticed that anti-miR-CTL relieved the ATM expression to some extent, which might be caused by the nonspecific binding of anti-miR-CTL to the endogenous miR-421 (Fig. 4 F and G).

Fig. 4.

miR-421 is up-regulated by N-Myc overexpression in HeLa cells. (A) Chromosomal location of miR-374b/miR-421 cluster on chromosome Xq13, sharing the same promoter. The promoter region (1 kb), containing an E-box (5′-CACGTG-3′), was cloned into luciferase construct pGL3-basic to create pGL3-PR421 and drives the transcription of firefly luciferase (Luc). (B) Luciferase activity of the miR-421 promoter. Luciferase constructs [pGL3-PR421 and unmodified construct (pRL)-CMV] were cotransfected, with vector (Vec) or N-Myc, into HeLa cells. Firefly luciferase activity was measured 24 h and 48 h after incubation and normalized to Renilla luciferase activity. (C) Real-time PCR of endogenous miR-421expression in HeLa cells transiently transfected with vector or N-Myc. Data were normalized to RNU66. (D) Immunoblot of ATM expression in HeLa cells transiently transfected with increasing amounts of N-Myc (using vector to compensate for equal amounts of total DNA). The fold change in ATM protein expression is shown below the blot. (E) ELISA measurement of ATM concentrations in HeLa cells transiently transfected with N-Myc. The asterisk indicates significant inhibition of ATM by N-Myc overexpression. (F) ELISA to determine ATM concentration in HeLa cells transiently transfected with the indicated DNA constructs (vector or N-Myc) and anti-miR-CTL (50 nM) or anti-miR-421(50 nM) inhibitors. (G) Immunoblot of ATM expression in HeLa cells transiently transfected with indicated DNA constructs (vector or N-Myc) and anti-miR-CTL (50 nM) or anti-miR-421(50 nM) inhibitor. Only the top band corresponds to ATM.

N-Myc/miR-421/ATM Pathway in Neuroblastoma Cells.

The N-Myc gene is frequently amplified in human neuroblastoma cells and is used as a prognostic marker for neuroblastoma (23, 24). To further explore the N-Myc/ATM relation, we examined ATM expression in seven human neuroblastoma cell lines: Four cell lines (CHLA-134, CHLA-136, LA-N-1, and LA-N-5) are N-Myc amplified, whereas the other three (CHLA-15, CHLA-90, and CHLA-255) are not N-Myc amplified. We noted a low level of N-Myc expression in CHLA-90 cell lines compared with the other two cell lines, CHLA-15 and CHLA-255, with undetectable N-Myc expression (Fig. 5A). We found that the ATM expression levels were significantly lower in the four N-Myc-amplified cell lines compared with those of three N-Myc-nonamplified cell lines (Fig. 5A), suggesting that N-Myc might negatively regulate ATM expression through miR-421. To confirm the interaction of N-Myc, miR-421, and ATM in neuroblastoma cells, we selected LA-N-1 (N-Myc⁺) and CHLA-255(N-Myc⁻) for the following experiments. i) Chromatin immunoprecipitation (ChIP) showed that the in vivo binding of N-Myc to miR-421 promoter only occurred in the N-Myc-amplified LA-N-1 cells and not in the N-Myc-nonamplified CHLA-255 cells (Fig. 5B). ii) Consistent with the in vivo binding of N-Myc, endogenous miR-421 expression was up-regulated (∼2-fold) in LA-N-1 cells (Fig. 5C). We also examined miR-421 expression in the other five neuroblastoma cell lines. The miR-421 levels were significantly higher in the four N-Myc-amplified cell lines (Fig. S3A). We noticed that the miR-421 level in CHLA-90 was higher than that in the other two N-Myc-nonamplified cell lines CHLA-15 and CHLA-255 (Fig. S3A). This might be caused by the low-level expression of N-Myc in CHLA-90 (Fig. 5A). A similar expression pattern for miR-374b was observed in these neuroblastoma cell lines (Fig. S3B), supporting a model that the miR-421 and miR-374b cluster is driven by the same promoter (Fig. 4A). iii) Treatment of LA-N-1 cells with AMO-ATM, which is complementary to the miR-421 binding sites at ATM 3′UTR, and with anti-miR-421 inhibitor, which is complementary to miR-421, led to an increase in ATM expression (Fig. 5D). As expected, AMO-ATM treatment did not change the miR-421 expression level, whereas anti-miR-421 inhibitor down-regulated miR-421 expression (Fig. 5E), suggesting two different mechanisms for AMO-ATM and anti-miR-421 on the abrogation of miR-421-mediated down-regulation of ATM expression. Finally, the increase of ATM expression by AMO-ATM was further confirmed by the flow cytometry phospho-SMC1 (FC-pSMC1) assay, which was recently developed to measure ATM kinase activity in A-T patients and carriers (25). As shown in Fig. 5F, treatment of LA-N-1 cells with AMO-ATM caused a post-IR shift in pSMC1, whereas treatment of CHLA-255 cells showed no change in the post-IR pSMC1 shift. These observations are compatible with the model that AMO-ATM could increase ATM expression in N-Myc-amplified neuroblastoma cells and are also consistent with the immunoblot results of ATM expression as shown in Fig. 5D.

Fig. 5.

N-Myc negatively regulates ATM via miR-421 in neuroblastoma cells. (A) Immunoblot of ATM expression in N-Myc-amplified (CHLA-134, CHLA136, LA-N-5, and LA-N-1) or -nonamplified (CHLA-15, CHLA-90, and CHLA-255) neuroblastoma cell lines. We observed some N-Myc expression in CHLA-90, although it is an N-Myc-nonamplified cell line; ATM expression was relatively lower in this cell line when compared with CHLA-15 or CHLA-255. A-T lymphoblastoid cells (AT-LCL) and WT lymphoblastoid cells (WT-LCL) are negative and positive controls, respectively, for ATM expression. In total, 100 μg of total protein for all neuroblastoma cells and only 25 μg of total protein for AT-LCL and WT-LCL were loaded; SMC1 served as a loading control. (B) ChIP PCR assay detects the in vivo binding of N-Myc protein to the miR-421 promoter DNA. A PCR fragment of expected size (246 bp) was seen in the N-Myc-amplified (amp.) LA-N-1 cells immunoprecipitated with the specific anti-N-Myc antibody (lane 5) but not without antibody or with nonspecific mouse IgG (lanes 2 and 3). No signal was seen in the N-Myc-nonamplified CHLA-255 cells immunoprecipitated with no antibody, nonspecific mouse IgG, or specific anti-Myc antibody (lanes 7–9). PCR with input DNA was used as a positive control. (C) Real-time PCR of endogenous miR-421 in the N-Myc-amplified LA-N-1 cells and the N-Myc-nonamplified CHLA-255 cells. RNU66 was used as an internal control. (D) Immunoblot of ATM expression in CHLA-255 and LA-N-1 cells treated with AMO-scram (4 μM) or AMO-ATM (4 μM) for 5 days or in L-AN-1 cells transfected with anti-miR-CTL (100 nM) or anti-miR-421 inhibitor (100 nM) for 96 h. The fold change in ATM expression is shown below. (E) Real-time PCR of miR-421 expression in LA-N-1 cells treated with AMO-scram (4 μM) or AMO-ATM (4 μM) for 5 days or transfected with anti-miR-CTL (100 nM) or anti-miR-421 (100 nM) inhibitor for 4 days. RNU66 was used as an internal control. (F) FC-SMC1 detection of IR-induced ATM-dependent phosphorylation of SMC1 in AMO-treated neuroblastoma cells. LA-N-1 and CHLA-255 cells were treated with AMO-scram (4 μM) or AMO-ATM (4 μM) for 5 days and subjected to 10-Gy IR. The pSMC1 level is indicated by the fluorescence intensity. The filled peaks represent the cells without IR, and unfilled peaks represent post-IR cells. This panel is representative of three independent experiments. (G) Linear signaling pathway in which N-Myc up-regulates miR-421 expression and miR-421, in turn, down-regulates ATM expression by targeting its 3′UTR.

Because c-Myc shares a conserved E-box binding site (5′-CACGTG-3′) with N-Myc (Fig. 4A), we were prompted to determine whether c-Myc functions in a manner similar to N-Myc in up-regulating miR-421 expression. As shown in Fig. S4A, cotransfection of c-Myc with the miR-421 promoter construct into HeLa cells resulted in a significant increase in miR-421 promoter-driven luciferase activity, as did cotransfection of N-Myc. Endogenous miR-421 expression in HeLa cells was similarly increased ∼1.5-fold after transfection of c-Myc (Fig. S4B).

Discussion

Taken together, our experiments suggest a previously undescribed mechanism of ATM regulation in which a noncoding small RNA, miR-421, down-regulates ATM expression through targeting ATM 3′UTR. This substantially expands our understanding of ATM functions in cellular physiology, such as cell cycle checkpoint, radiosensitivity, and other ATM-mediated cellular functions. For example, microRNA profiling study has revealed that miR-421 is up-regulated in germinal center centroblast B cells (26), where physiological DNA damage occurs frequently because of somatic hypermutation and class switch recombination (27). The miR421-mediated ATM down-regulation in centroblasts might contribute to the escape of centroblast B cells from DNA damage-induced cell cycle checkpoints and allow centroblasts to develop into memory B cells or plasma cells. A recent report corroborates this concept in which ATR (ATM and Rad3-related) kinase is transiently silenced by a transcription repressor Bcl-6 in germinal center B cells (28). Interestingly, miR-421 expression is also up-regulated in diffuse large B-cell lymphoma cell lines (29), suggesting that this newly identified miR421–ATM interaction might be involved in the progression of diffuse large B-cell lymphoma. It is known that about 10% of cases have overexpression of c-Myc, a result of the c-myc translocation into the Ig locus (27).

We have established that miR-421 expression is up-regulated by the transcription factor N-Myc, establishing a linear signaling pathway (N-Myc → miR-421 → ATM) in such a manner that the oncogene N-Myc negatively regulates the tumor suppressor ATM (Fig. 5G). Because the ATM-driven DNA damage response is thought to be a physiological barrier in early human tumorigenesis (30 –33), our findings add that miR421-mediated ATM down-regulation may contribute to N-Myc-induced tumorigenesis in neuroblastoma. The finding that the up-regulation of miR-421 can alter the cellular radiosensitivity suggests that treatment of proliferating cancer cells with miR-421-inducing agents might sensitize them for radiotherapy. Conversely the finding that exposure of neuroblastoma cells to AMO-ATM increases ATM expression implies that AMO-ATM holds therapeutical potential for N-Myc-amplified neuroblastomas, perhaps by enhancing ATM-dependent apoptosis in response to DNA damage (34, 35) or driving nondividing differentiated neuronal cells to reenter S-phase (36). Lastly, the suppression of ATM by miR-421 introduces two possible pathogenetic mechanisms for A-T: A mutation in the ATM 3′UTR might enhance the binding of miR-421, or a mutation of miR-421 might result in miR-421 overexpression, both leading to the down-regulation of ATM expression. Such disease-causing mutations of microRNA-binding sites in the 3′UTR of the target genes have been reported (37). However, no such mutations have been observed to date in A-T patients. Our findings also suggest that miR-421 could function as a modifier gene, contributing to the A-T phenotype and perhaps to the variability of disease onset and progression.

Materials and Methods

Cell Culture, miRNA Precursors, miRNA Inhibitors, AMO and Transfection.

Neuroblastoma cell lines LA-N-1 and LA-N-5 were cultured in RPMI 1640 with 15% (vol/vol) FBS and streptomycin/penicillin, and CHLA-15, CHLA-90, CHLA-134, CHLA-136, and CHLA-255 were cultured in Iscove’s Modified Dulbecco’s Medium with 15% (vol/vol) FBS and streptomycin/penicillin. The precursor miR-421, pre-miR-CTL, anti-miR negative control 1, and anti-miR-421 inhibitor were purchased from Applied Biosystems. Antisense AMO was synthesized based on the ATM 3′UTR target sequence and conjugated with nonpeptide chemicals that are used to deliver AMO to cells (Gene-Tools). The sequence of AMO-ATM is 5′-ATCAACAGATATAAACAGCAGG. A standard control AMO (AMO-scram) was also purchased from Gene-Tools. N-Myc and c-Myc plasmids were obtained from Origene and Open Biosystems, respectively. All transfections were done with Lipofectamine 2000 (Invitrogen) according to the provided protocols. The M4 lentiviral vector expressing miR-421 was generated by standard methods as detailed in SI Text.

RNA Extraction and Real-Time Quantitative PCR.

Total RNA from cultured cells was extracted by the mirVana miRNA isolation kit (Applied Biosystems). TaqMan microRNA expression assays (Applied Biosystems) were used to quantitate mature miR-421 expression according to the provided protocol. RNU66 or U6 expression assay was used as an internal control for miR-421 expression. ATM mRNA quantification were measured by real-time PCR based on TaqMan Gene Expression Assays (Applied Biosystems), as previously described (38). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA was used as an internal control to normalize ATM mRNA level. Real-time PCR quantitation of transcripts was expressed as ATM/GAPDH ratios.

Luciferase Reporter Assays and Transcription Factor Analysis.

Cells were transfected with appropriate reporter vectors and then harvested and lysed for luciferase assays by a Dual-Glo assay kit (Promega) according to the manufacturer’s protocol. The detailed protocol is described in SI Text.

The transcription factor binding site analysis was done using the CONSITE database. A 2-kb genomic sequence upstream of miR-421 was used as the analyzing template, and the cutoff value for transcription factors was set to 99%; N-Myc transcription factor was the top candidate.

ATM-ELISA.

An ELISA was used to determine the relative ATM expression in cells and performed as previously described (39). More details on the ATM-ELISA are provided in SI Text.

BrdU Incorporation Assay.

To analyze the S-phase checkpoint, cells taken at 70% confluence were irradiated with the indicated dose and incubated for 20 h. BrdU was added to cells and incubated for 2 h. Cells were collected by trypsinization and centrifugation. Cells were subject to the BrdU flow staining according to the manufacturer’s protocol for BrdU Flow Kits (BD Pharmingen). Three independent experiments were performed.

Clonogenic Survival Assay and Propidium Iodide-Staining Cell Death.

MiR-421-expressing stable and control shRNA cells were plated at 500 cells per well onto a six-well dish in triplicate and then incubated for 24 h to allow settling. Cells were treated with a series of IR doses (0, 1, 2, and 5 Gy) and grown for 2 weeks before staining with 1% crystal violet. Clumps containing more than 50 cells were scored as “colony-positive” wells and counted by the Quantify One program in the VersaDoc Imaging System (Bio-Rad). To generate a radiation survival curve, the surviving fraction at each radiation dose was normalized to that of a nonirradiated control. All experiments were repeated at least twice. For IR-induced cell death, cells were treated with 10-Gy radiation and stained with propidium iodide after 48 h of incubation to assess the number of dead cells. Samples were analyzed by a FACScan Analytic Flow Cytometer (Becton Dickinson). Three independent experiments were done. Cell death was normalized to nonirradiated control cells.

FC-pSMC1 Assay and ChIP Assay.

An FC-pSMC1 assay was performed as previously described (25), and a ChIP assay was performed as previously described (40). More detailed protocols for Fc-pSMC1 and ChIP assays are provided in SI Text.

Statistics.

The Student’s t -test was used to evaluate the significant difference of two groups of data in all the pertinent experiments. A P value <0.05 (using a two-tailed paired t test) was thought to be significantly different for two groups of data.