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The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia

The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia Background: Recent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis. Methods: The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML st patients in 1 complete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA-β-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1. Results: We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2 -deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells. Conclusions: Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression. Keywords: miR-370, FoxM1, AML, Cellular senescence Introduction microRNAs (miRNAs), small (~22 nucleotide), single- Acute myeloid leukemia (AML) is a heterogeneous stranded noncoding RNAs, are a novel class of biological group of neoplastic haematopoietic diseases character- molecules. Their genes may either give rise to single ized by proliferation and maturation arrest of myeloid miRNAs, or contain several miRNAs in one transcrip- blasts in bone marrow and blood [1]. The long-term tional unit as miRNA clusters [3]. miRNAs post- overall survival (OS) rate for AML patients under the transcriptionally repress gene expression by recognizing age of 60 years and 60 years or older is 30–40% and complementary target sites in the 3 untranslated region under 10%, respectively [2], which remains a challenge. (UTR) of target mRNAs [4,5]. miRNAs have been impli- Thus, it is urgently needed to search for new targets for cated in a large variety of biological processes, including molecularly designed therapies. cell cycle progression, apoptosis, differentiation and haematopoiesis [6-10], and thereby play important roles in many pathological processes, including malignant * Correspondence: [email protected] transformation [11,12]. More than 50% of miRNA genes Equal contributors 1 are located in cancer-associated genomic regions or in Department of Hematology, Qilu Hospital, Shandong University, No.107, fragile sites, and both oncogenic and tumor suppressive Wenhua Xi Road, Jinan 250012 Shandong, P. R. China Full list of author information is available at the end of the article © Zhang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang et al. Molecular Cancer 2012, 11:56 Page 2 of 11 http://www.molecular-cancer.com/content/11/1/56 functions have thus far been ascribed to specific miR- treatment and after a complete remission and we found NAs [13]. Moreover, miRNAs have emerged as critical a lower miR-370 level at diagnosis while at least 2.1-fold regulators of hematopoiesis and their aberrant expres- increase in miR-370 expression after CR (Figure 1B). sion has been associated with the pathogenesis of leukemia [14]. Functional validation of deregulated miR- Changes in proliferation and cellular senescence of NAs in hematopoeisis has been shown for several miR- leukemic cells mediated by altered miR-370 expression NAs [15]. Distinctive patterns of increased expression We then explored the biological function of miR-370 in and/or silencing of multiple miRNAs have been asso- leukemic cells. Cells were transfected with precursors to ciated with specific cytogenetic and molecular subsets of miR-370 and miR-370 inhibitor to enhance and decrease AML [16]. miR-370 has been noted to be down- mature miR-370 expression, respectively. Transfection regulated in papillary thyroid carcinoma, colorectal can- with the miR-370 precursor increased mature miR-370 cer [17] and malignant cholangiocytes [18], but evidence expression 114.5 ± 5.70: 1 ± 0.12 (p < 0.05) and 59.8 ± of a biological role for this miRNA in AML has not been 6.90: 1 ± 0.24 (p < 0.05) (pSilencer-miR vs pSilencer) reported. In the present study, we sought to define the times higher in HL60 and K562 cells, respectively role of miR-370 in AML by investigating its expression (Figure 2A). Overexpression of miR-370 decreased cell and biological function in leukemic cell lines and blast proliferation (Figure 2C and Additional file 1) (pSilencer cells from patients with de novo AML. vs pSilencer-miR: HL60: 88 ± 15 vs 11 ± 4, p < 0.01; K562: 49 ± 5 vs 18 ± 5, p < 0.01). On the other hand, Results transfection with the miR-370 inhibitor suppressed mature Down-regulation of miR-370 expression in BM blasts from miR-370 expression to 31% ± 0.04 (p < 0.05) and 58% ± de novo AML patients 0.05 (p < 0.05) lower in HL60 and K562 cells, respectively We analyzed miR-370 expression in BM samples from (Figure 2B). The decline in miR-370 expression was 48 de novo AML patients at diagnosis time using qRT- coupled with enhanced cell proliferation (Figure 2C) (pSu- PCR. As shown in Figure 1A, the miR-370 level in per vs pSuper-miR-inhibitor: HL60: 56 ± 7 vs 72 ± 6, p < patients’ samples was significantly reduced (P < 0.01, 0.05; K562: 66 ± 12 vs 93 ± 7, p < 0.05). t test) compared to that from healthy controls, while fol- The above result suggests that miR-370 suppresses lowing acquisition of CR in the induction chemotherapy, proliferation of HL60 and K562 cells. We further wanted miR-370 expression level restored to 0.82 fold of con- to define the mechanism behind miR-370 overexpres- trols. There was no association between the presence of sion-mediated proliferation inhibition. We suspected mature miR-370 and age, gender, blast percentage or that miR-370 might trigger cellular senescence program. FAB subtypes (data not shown). In six patients, BM sam- Senescence-associated β-Gal staining, a specific marker ples were available both at diagnosis time prior to for senescent cells [19], was thus performed. A positive Figure 1 Downexpression of miR-370 in de novo AML patients. (A) miR-370 expression by qRT-PCR in 48 de novo AML patients, 40 AML st patients of 1 CR and 21 healthy controls. (B) Restoration of miR-370 expression in six patients after complete remission achievement in induction chemotherapy. The results are shown as miRNA expression after normalization with U6 and -ΔCt calculations. Zhang et al. Molecular Cancer 2012, 11:56 Page 3 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 2 (See legend on next page.) β-Gal staining was observed in the two cell lines trans- Up-regulation of miR-370 expression mediated fected with miR-370 precursors (Figure 2D) [pSilencer by 5-Aza-CdR vs pSilencer-miR (% of β-Gal-positive cells): HL60: 3 ± 1 DNA methylation is an epigenetic modification that reg- vs 28 ± 3, p < 0.01; K562: 8 ± 3 vs 40 ± 1, p < 0.01;]. ulates gene expression. Aberrant DNA methylation has Zhang et al. Molecular Cancer 2012, 11:56 Page 4 of 11 http://www.molecular-cancer.com/content/11/1/56 (See figure on previous page.) Figure 2 Suppression of AML cell proliferation and induction of cell senescence by overexpression of miR-370 in vitro while anti-miR-370 expression increases cellular ability of proliferation. (A) miR-370 expression in AML cell lines and its overexpression using a specific miR-expressing pSilencer vector. The cells were transfected with either miR-370 precursors or with control precursor and harvested after 48 hours. qRT-PCR was applied to detect mature miR-370 level. (B) The depletion of miR-370 expression by a specific miR-370-inhibiting pSuper plasmid. The method was similar as above. (C) Alterations in foci formation after different treatments in AML cells. The cells were first treated as above and the efficiently changed miR-370 expression was verified in (A) and (B). For clonogenic assays, 5,000 cells/well (in 6-well plates) were incubated for 14 days and the foci number was counted. (D) Senescence onset of miR-370-overexpressed AML cells. β-Gal staining was performed and % of positive cells was calculated. been implicated in many cancers [20]. Global hypo- (Figure 4C-D). There was a >2-fold increase in expres- methylation or aberrant hypermethylation of gene pro- sion of FoxM1 in HL60 and K562 cells after transfection moter CpG islands result, respectively, in tumor cell of miR370 inhibitor plasimids (Figure 4E). 5-aza-CdR genomic instability and gene silencing, particularly of significantly reduced the expression of FoxM1 in both tumor suppressor genes [17]. Interestingly, the chromo- HL60 and K562 cells (Figure 3 B-C). These changes somal location of miR-370 on chromosome 14q32.31 were similar to those observed with miR-370 overex- has been shown to be regulated by DNA methylation, or pression. Taken together, FoxM1 is a target of miR-370. deleted by loss of heterozygosity [14,21] or by hyper- methylation of an CpG island 200 bp upstream in the Overexpression of FoxM1 in de novo AML patients mother allele [22]. Treatment with 5 μM 5-aza-CdR, a FoxM1, a master regulator of mitotic gene expression, is DNA methylation inhibitor, for 72 hours, substantially required for cell proliferation and its inhibition leads to (>2.0-fold) and significantly (P < 0.05) increased the ex- reduction in anchor-independent growth and tumori- pression of miR-370 in both HL60 and K562 cells genesis of cancer cells [27]. As we have verified that (Figure 3A) and decreased cell proliferation (Figure 3D) FoxM1 is a target for miR-370, we then sought to probe (control vs CdR: HL60: 24 ± 4 vs 7 ± 2, p < 0.01; K562: its role in AML. The tumor specimens from forty-eight 152 ± 5 vs 78 ± 5, p < 0.001). de novo AML patients and forty AML patients in 1st CR and twenty-one healthy controls were analyzed for Identification of FoxM1 as a target for miR-370 FoxM1 mRNA expression using qRT-PCR. Patient char- To further elucidate the mechanism by which miR-370 acteristics are described in Table 1. The FoxM1 tran- affected cellular senescence and proliferation, we next script level in AML patients was found 21.47-fold higher screened for potential targets of miR-370 using four tar- than that in controls, while following acquisition of CR get prediction programs with different algorithms: in the induction chemotherapy, FoxM1 expression level DIANA-MicroT [23], TargetScan [24], Miranda [25] and reduced to 1.75 fold of controls. (Figure 5 A, C), which PicTar [26]. All potential targets predicted by more than was negatively correlated with miR-370 levels. There was one of these programs were identified. We selected the a highly significant difference in FoxM1 expression be- forkhead box M1 (FoxM1) for further study because of tween AML samples, CR samples and healthy controls its well-characterized role in tumor biology. The FoxM1 (qRT-PCR, One-Way ANOVA, p < 0.01). In six patients, gene has a 249-bp 3 UTR region that presents a 7-mer BM samples were available both at diagnosis time prior binding site for miR-370 (Figure 4A). to treatment and after a complete remission and we First, we made the luciferase reporter constructs con- found a higher FoxM1 level at diagnosis while a signifi- taining the miR-370 recognition sequence from the 3 - cant decrease in FoxM1 expression after CR except one UTR of FoxM1 inserted downstream of the luciferase sample (Figure 5B). There was no clear association be- gene. Transfection with miR-370 precursor decreased re- tween the presence of FoxM1 mRNA and age, gender, porter activity in K562 cells (Figure 4B), which strongly tumor burden or FAB subtypes (data not shown). More- indicates that FoxM1 is a target for miR-370. Next, the over, BM materials from 8 de novo AML patients, 8 studies were repeated with random mutations in the rec- patients in 1st CR and 5 healthy controls randomly ognition sequence (Figure 4A), which resulted in aboli- chosen from our AML patient pool were used to deter- tion of the reporter activation by miR-370 precursor mine the mRNA expression level of c-Myc, hTERT, kip1 (Figure 4B). Finally, we assessed the effect of miR-370 p27 and skp2, all of which were the target genes of expression on FoxM1 expression. Transfection of HL60 FoxM1, using real-time PCR method. We found that the and K562 cells with miR-370 precursor resulted in lower transcript levels of c-Myc, hTERT and skp2 in AML expression of FoxM1 after 48 hours (Figure 4C-D). Con- patients were respectively 9.64, 3.76 and 3.14-fold higher comitant with decreased FoxM1 expression, there was than those in controls, while following acquisition of reduction of its downstream target c-myc and skp2 CR, all of them reduced almost to the same levels of Zhang et al. Molecular Cancer 2012, 11:56 Page 5 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 3 Expression of miR-370 in AML cells (HL60 and K562) and the effect of 5-aza-2’deoxycytidine (5-Aza-CdR). AML cells were plated in 6-well plates, treated with 5 μM 5-Aza-CdR or PBS and harvested after 72 hours. The expression of mature miR-370 was assessed using real-time PCR. (A) 5-aza-CdR increases the relative expression of miR-370 in AML cells. Data represent mean ± s.e. from three separate determinations. *p < 0.05 compared with respective controls. (B) 5-aza-CdR decreases the relative mRNA level of FoxM1 in AML cells. *p < 0.05. (C) 5-aza-CdR decreases the protein level of FoxM1 in AML cells. (D) Diminished foci formation of 5-aza-CdR treated AML cells. kip1 controls. On the contrary, the expression level of p27 regulated and coupled with the accumulation of the kip1 in AML patients is only 40% of that in controls, while CDK inhibitor p27 (Figure 5 D-E). restored after CR (Additional file 2). Collectively, the FoxM1 gene is aberrantly activated in AML and is required for sustained proliferation of the Defective foci formation by depletion of FoxM1 in AML cancer cells. cell lines Then, we employed the FoxM1-specific siRNA to knock Discussion down FoxM1 expression in AML cell lines and the cells In the present study, we explored the expression and were then assessed for their clonogenic capacity. Effi- role of miR-370 in AML. Our findings show a down- cient silence of FoxM1 expression in these cells was regulation of miR-370 in blasts from patients with de verified using qRT-PCR and Western blot analyses novo AML. Moreover, we identified FoxM1 as a target (Figure 5 D-E). Compared to the control cells, the for miR-370 and restored expression of miR-370 reduced FoxM1-knocked down cells exhibited significantly the level of FoxM1. In hematological malignancies a link diminished foci formation (Figure 5F) (Controls vs with miRNA was initially described in chronic lympho- FoxM1 siRNA: HL60: 19 ± 3 vs 11 ± 2, p < 0.05; K562: cytic leukemia (CLL) by Calin et al. [28]. A cluster of 33 ± 5 vs 5 ± 2, p = 0.001). Following FoxM1 depletion, two miRNAs, miR-15a, and miR-16, was found to be its target genes c-myc, skp2 and hTERT were also down- located within the deleted region at 13q14, and down- Zhang et al. Molecular Cancer 2012, 11:56 Page 6 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 4 Identification of FoxM1 as a target of miR-370. (A) Predicted site of miR-370 in the FoxM1 3’ UTR mRNA. (B) Luciferase activities were determined at 24 hours and were normalized by Renilla luciferase activity. K562 cells were plated in 24-well plates, and then transfected with 0.05 μg of a Renilla luciferase expression construct pRL-TK and 0.5 μg of the pGL3-FoxM1-wt-luc or pGL3-FoxM1-mut-luc firefly luciferase expression construct, along with either miR-370 precursor or control precursor. Luciferase assays were performed after 24 hours using the dual luciferase reporter assay system. The expression of firefly luciferase activity was normalized to that of renilla luciferase activity for each sample. A decrease in relative firefly luciferase activity in the presence of miR-370 indicates the presence of a miR-370 modulated target sequence in the 3’- UTR of FoxM1. Data represents mean ± s.e. from three separate experiments. *P < 0.05. (C) and (D) The expression of FoxM1 and downstream genes were regulated by miR-370 overexpression at both transcription level and translation level. Western blots were performed and sequentially kip1 probed with antibodies against FoxM1, c-Myc and p27 , β-actin was a loading control. Efficient miR-370 overexpression was verified in Figure 2 (A). (E) Increase in FoxM1 protein level after anti-miR-370 treatment. Efficient miR-370 depletion was verified in Figure 2 (B). regulated in the majority (70%) of CLL samples. Notably, with ALL on the basis of 21 up-regulated and 6 down- miR-15a and miR-16-1 down-regulation contribute to regulated miRNAs, among which four of them (let-7b, malignant transformation by up-regulating BCL2 [29]. miR-128a, miR-128b and miR-223) were the most dis- Recently, involvement of miRNA in AML has been criminatory [31]. According to Amanda Dixon-McIver, documented identifying that miRNA expression profiles compared with other major cytogenetic subgroups of are AML subtype-specific and suggesting a pathogenetic AML, APLs bearing the t(15;17) translocation were role of miRNA in AML. For example, Mi and colleagues characterised by the up regulation of 7 miRNAs tran- [30] showed distinct miRNA signatures between acute scribed from genes located at the 14q32 region. The set lymphoblastic leukemias (ALLs) and AMLs. In that includes miR-127, miR-154, miR-154*, miR-299, miR- study, patients with AML could be separated from those 323, miR-368, and miR-370. Two other groups reported Zhang et al. Molecular Cancer 2012, 11:56 Page 7 of 11 http://www.molecular-cancer.com/content/11/1/56 Table 1 Patient Characteristics cancer development [35]. For instance, miR-127 has Characteristic De novo AML patients been shown to be down regulated or silenced in cancer st AML patients in 1 CR cells, whose expression is correlated with the methyla- Patients,no. 48 40 tion and acetylation status of its promoter. Inhibition of methylation and histone deacetylation in these cancer Sex,male/female,no.(%) 31(65)/17(35) 24(60)/16(40) cells causes over expression of miR-127 and related Median age,y(range) 41(14–74) 41(14–68) down regulation of the target BCL6, a bona fide pro- WBC count (10 /L) tooncogene [36]. We hypothesize that miR-370 also Median 46.3 6.3 acts as a tumor suppressor in AML, as in papillary Range 0.8-354.9 2.6-10.2 thyroid carcinoma, colorectal cancer and malignant Hemoglobin (g/dL) cholangiocytes. The comparison of the leukemia sam- ples with healthy controls highlighted the differential Median 7.7 11.8 expression of miR-370. Following the treatment with Range 4.1-13.2 8.3-16.1 5-aza-2 -deoxycytidine, there is a significant enrich- Platelet count (10 /L) ment for miR-370 in AML cell lines, which indicated Median 58 243.3 that hypermethylation may contribute to reduction of Range 2-319 76-384 miR-370. BM blasts (%) Cancer therapy has traditionally relied on cytotoxic treatment strategies on the assumption that complete Median 69.1 2.1 cellular destruction of tumors optimizes the potential for Range (24–96) (0–5) patient survival. Although these approaches produce FAB subtype,n/(%) complete cell death within a tumor, they also can cause M2 9/(19) 6/(15) severe side effects in patients [37]. Recently, a promising M3 11/(23) 20/(50) approach to preventing continued tumor growth is M4 11/(23) 8/(20) therapy-induced senescence (TIS) [38]. Senescent cells remain viable and metabolically active but are perman- M5 15/(31) 5/(12.5) ently growth arrested [39]. Evidence has recently accu- M6 2/(4) 1/(2.5) mulated that cellular senescence is a potent barrier to cancer development. Our results indeed demonstrated independently that miR-155 was up-regulated in AML that senescence occurred in most of AML cells treated patients with FLT3-ITD, suggesting that this miRNA with miR-370 overexpressing plasmid, which was con- contributes to the highly proliferative phenotype of this comitant with their diminished clonogenic capacity. molecular subset of AML [31-33]. In the present study, Given a critical role for senescence induction in tumor we haven’t got the conclusion that the expression level suppression and therapeutic efficacy of cancer treat- of miR-370 is with AML subtype-specificity, which may ment, the present findings have important biological be due to the limited number of primary AML patients and clinical implications. All these results suggest that enrolled in our study. downregulation of miR-370 may be another mechanism DNA methylation is an epigenetic modification that involved in the pathology of AML and therefore, could can regulate gene expression. Aberrant DNA methyla- be used as a diagnostic marker and therapeutic target tion has been implicated in many cancers [21]. miR-370 in AML. and many other miRNAs are organised in clusters to- We have also analyzed the correlation between miR- gether on chromosome 14q32 [34]. This miRNA cluster 370 expression and FoxM1 mRNA expression in 48 de acts as imprinted non-coding RNA (ncRNA) genes, novo AML samples. Consistent with the cell line data, which are mono-allelically expressed in a parent-of- FoxM1 was enriched in the primary blasts mRNAs that origin manner (their expression is restricted to either inversely correlated to miR-370 expression levels. This the maternal or the paternal allele). Interestingly, as for result was also observed at the protein level in a few pri- this miRNA cluster, which is of maternal origin, its mary AML samples. However, further studies using large imprinted expression is regulated by an intergenic differ- numbers of primary AML samples will be needed to entially methylated region (DMR) located ~ 200 kb up- confirm this interaction. stream from the miRNA cluster [24]. Hypermethylation of DMR causes silence of this miRNA cluster, including Conclusion miR-370. It has been suggested that miRNAs in this re- We demonstrate that miR-370 is a tumor suppressive gion act as tumour repressor genes and that changes in factor by targeting multiple critical oncogenic pathways. the methylation status of their promoters could trigger Restoring miR-370 expression downmodulates FoxM1, Zhang et al. Molecular Cancer 2012, 11:56 Page 8 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 5 Overexpression of FoxM1 in de novo AML patients and defective foci formation induced by depletion of FoxM1 in AML cell lines. (A) and (C) Expression of FoxM1 mRNA and protein in de novo AML patients, AML patients of 1st CR and controls, were determined using qRT-PCR and Western blot respectively. (B) Decrease of FoxM1 expression in six patients after complete remission achievement in induction chemotherapy. (D) and (E) FoxM1 depletion triggers concomitant down-regulation of c-myc, hTERT and skp2 coupled with the accumulation of kip1 the CDK inhibitor p27 in AML cells. The cells were transfected with either control or FoxM1 siRNA and harvested after 72 hours. qRT-PCR and western blot were applied to detect mRNA and protein levels, respectively. (F) Diminished foci formation of FoxM1-depleted AML cells. The cells were first treated as above and the efficient FoxM1 knock down was verified in (D) and (E). induces senescence, and dampens cell growth in AML presentation and morphologic criteria of the French- cells, thereby suggesting miRNA-based therapy as a American-British (FAB) Classification. The study was novel approach to increase response in AML. approved by the local ethics committee. Patients’ BM samples were collected between April 2008 and Septem- Materials and methods ber 2011 at the Department of Hematology, Qilu Hos- Patients and bone marrow samples pital, Shandong University, Jinan, China. Mononuclear Forty-eight newly diagnosed AML patients (31 male and cells were isolated using Ficoll-Hypaque density gradient 17 female; median age at diagnosis 41; range 14–74), centrifugation, and then stored at −80°C until use. All st forty AML patients in 1 complete remission (CR) and patients and healthy controls were tested for miR-370 twenty-one healthy controls were enrolled in this study. and FoxM1 mRNA levels in their BM cells. Among Diagnosis of AML was established according to clinical those AML patients, six were analyzed for miR-370 and Zhang et al. Molecular Cancer 2012, 11:56 Page 9 of 11 http://www.molecular-cancer.com/content/11/1/56 FoxM1 levels in their bone marrow samples at both Western blot diagnosis and complete remission. Total cellular proteins were extracted from cultured cells or BM samples. Proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The mem- Cell lines and culture conditions branes were probed with the specific antibodies against Human AML cell lines HL60 and K562 were cultured at FoxM1, p27, c-MYC (Santa Cruz Biotechnologies, Santa 37°C, 95% air, 5% CO in RPMI 1640 containing 10% Cruz, CA, USA), followed by anti-mouse or rabbit horse- heat-inactivated fetal bovine serum (FBS; Gibco, radish peroxidase–conjugated IgG and developed with Carlsbad, CA, USA), 100 μg/mL penicillin, and 50 μg/mL the enhanced chemiluminescence method (ECL). β-actin streptomycin. To assess 5-aza-CdR (Sigma, Santa Clara, served as a loading control. CA, USA) effects, cells were grown on 6-well plates, trea- ted with 5 μM 5-aza-CdR or cold phosphate-buffered sa- miR-expressing and miR-inhibiting plasmids line (PBS) controls for 72 h at 37°C, and then harvested To generate the miR-expressing pSilencer3.1-H1 neo for isolation of total mRNA or protein. vector, a fragment of 212 base pairs (bp) corresponding to the desired miRNA and the surrounding sequences RNA extraction and quantitative real-time PCR was amplified from human genomic sequence, adding a 0 0 Total cellular RNA in BM samples and in cells with dif- BamHI site and a HindIII site to the 5 and 3 ends re- ferent treatments was extracted using the Trizol (Invitro- spectively, using the polymerase chain reaction (PCR) gen, Carlsbad, CA, USA) according to the manufacturer’s with primers sense: 5 AAGGGATCCTACTTGAGG 0 0 protocol. cDNA was synthesized using random primers GATGGGCGATA 3 and antisense 5 TCAAAGCTTC (N6) (Fermentas, St. Leon-Rot, Germany) and MMLV re- CCGAGCTCTGGTGTTAGAC 3 . We included a large verse transcriptase. The PCR primers used in the study portion of miRNA surrounding sequence in the attempt were as follows: sequences specific for FoxM1 mRNA: 5 - to allow correct processing of the miRNA to its mature 0 0 TGCAGCTAGGATGTGAATCTTC-3 (Forward) and 5 - form and to induce overexpression while preserving a GGAGCCCAGTCCATCAGAACT-3 (Reverse). Skp2: physiologic mechanism of miRNA production. miR-370 0 0 5 -GGACCTATCGAACTCAGTTAT-3 (Forward) and inhibitor sequences were synthesized as DNA oligonu- 0 0 kip 5 -CAGCCACCTGTACATGCTTT-3 (Reverse); p27 : cleotides; after annealing, were sticking ended and sub- 0 0 5 -ATGTCAAACGTGCGAGTGTCTAA-3 (Forward) and cloned into a pSuper vector. 0 0 5 -TTACGTTTGACGTCTTCTGAGG-3 (Reverse); hTERT: 0 0 0 5 -CGGAAGAGTGTCTGGAGCAA-3 (Forward) and 5 - Transfection 0 0 GGATGAAGCGGAGTCTGGA-3 (Reverse); c-MYC: 5 - Cells were incubated in 6-well plates (3.0× 10 /well and TACCCTCTCAACGACAGCAGCTCGCCCAACTCCT-3 1.0× 10 /well, respectively) overnight and were then (Forward) and 5 -TCTTGACATTCTCCTCGGTGTCC transfected with plasmid or siRNA using Lipofectamine GAGGACCT-3 (Reverse). The above primer pairs cross 2000 (Invitrogen, Carlsbad, CA, USA) according to the intron/exon boundaries; thus, the resultant PCR products manufacturer’s protocol. Chemical modified Stealth small do not represent genomic DNA contamination. β-actin interfering RNA (siRNA) targeting FoxM1 and control expression was used as a control for RNA loading and RT siRNA were bought from Invitrogen. The sequence for efficiency and amplified. Quantitative real-time polymerase the FoxM1 siRNA was 5 -GACAACUGUCAAGUGUAC chain reaction (qRT-PCR) was carried out in an ABI7000 CACUCUU-3 . sequence detector (Applied Biosystems, Foster City, CA, USA). Soft agar colony formation assay HL60 and K562 cells were resuspended in DMEM (Gibco, TaqMan qRT-PCR miRNA analysis Carlsbad, CA, USA) containing 20% heat-inactivated fetal Quantification of mature miRNAs was performed using bovine serum (FBS; Gibco, Carlsbad, CA, USA) with equal qRT-PCR with the TaqMan miRNA assay kit (Applied amount of either 0.3% agar (HL60 cells) or 0.5% agar Biosystems, Foster City, CA, USA) according to manufac- (K562 cells), and plated in 6-well plates at 5,000 per well turer’s instruction. Briefly, 10 ng of total RNA was re- on top of a 2 mL precast semisolid 1% agar underlayer as verse-transcribed (RT) with specific primers, subsequently described previously [40]. The number of colonies with 1.5 μL of RT product was used as template for real-time more than 50 cells was counted after two weeks. PCR. All real-time experiments were performed in tripli- cate. Data were normalized by the expression of small Senescence-associated β-Galactosidase nuclear RNA (snRNA) U6 and expressed either as rela- (SA -β-Gal) staining -ΔCt tive expression (2 ) or as fold change relative to con- SA -β-Gal staining was done as described [19,41]. Briefly, -ΔΔCt trol (2 ). the cells grown in 6-well plates were transfected with Zhang et al. Molecular Cancer 2012, 11:56 Page 10 of 11 http://www.molecular-cancer.com/content/11/1/56 pSilencer or pSilencer-miR-370. After 7 days, the cells were Acknowledgements This work was supported by the National Natural Science Foundation of rinsed with PBS once, fixed in 3% of formaldehyde for China (Nos. 81170514, 30971151, 81172354, 30972775 and 81001098), the 15 min, and incubated with freshly prepared SA -β-Gal Project Sponsored by the Scientific Research Foundation for the Returned staining solution at 37°C overnight. Overseas Chinese Scholars, State Education Ministry (The Project-sponsored by SRF for ROCS, SEM), Shandong Provincial Natural Science Foundation, China (Nos. ZR2009CM002, ZR2009CZ001, ZR2010HZ003 and ZR2012HQ003), Luciferase reporter vector Independent Innovation Foundation of Shandong University (IIFSDU, Nos. 2010TS074 and 2012TS106) and the National Basic Research Program of The precursor to miR-370 was synthesized and cloned in China (973 Program, 2012CB911202). pSilencer. Firefly luciferase reporter vectors with the intact putative miR-370 recognition sequence from the 3 -UTR Author details Department of Hematology, Qilu Hospital, Shandong University, No.107, of FoxM1 (pGL3-FoxM1-wt-3 -UTR) or with random 0 Wenhua Xi Road, Jinan 250012 Shandong, P. R. China. Department of mutations (pGL3-FoxM1-mut-3 -UTR) cloned down- 3 Biochemistry, Shandong University, Jinan, China. Department of stream of the firefly luciferase gene were constructed. Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, Jinan, China. Wild-type and mutant inserts were confirmed by sequen- 0 Department of Medicine, Division of Hematology and CMM, Karolinska cing. For the 3 UTR-luciferase assays, cells were co- University Hospital Solna and Karolinska Institutet, Stockholm, Sweden. transfected with 0.5 μg pGL3-FoxM1-wt or mut-3 -UTR Received: 21 April 2012 Accepted: 6 August 2012 construct, 4 μg of pSilencer or pSilencer-miR and 0.05 μg Published: 17 August 2012 pRL-TK Renilla luciferase expression construct using Lipo- fectamine 2000. Luciferase assays were performed 24 h References after transfection using the Dual Luciferase Reporter Assay 1. Estey E, Döhner H: Acute myeloid leukaemia. Lancet 2006, system (Promega,Madison,Wisconsin,USA). 368:1894–1907. 2. Estey EH: Treatment of acute myeloid leukemia. Haematologica 2009, 94:10–16. Statistical analyses 3. Altuvia Y, Landgraf P, Lithwick G, Elefant N, Pfeffer S, Aravin A, Brownstein MJ, Tuschl T, Margalit H: Clustering and conservation patterns of human The difference in miR-370 and FoxM1 mRNA expres- microRNAs. 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The tumor suppressive role of miRNA-370 by targeting FoxM1 in acute myeloid leukemia

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Copyright © 2012 by Zhang et al.; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology
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10.1186/1476-4598-11-56
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Abstract

Background: Recent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis. Methods: The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML st patients in 1 complete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA-β-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1. Results: We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2 -deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells. Conclusions: Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression. Keywords: miR-370, FoxM1, AML, Cellular senescence Introduction microRNAs (miRNAs), small (~22 nucleotide), single- Acute myeloid leukemia (AML) is a heterogeneous stranded noncoding RNAs, are a novel class of biological group of neoplastic haematopoietic diseases character- molecules. Their genes may either give rise to single ized by proliferation and maturation arrest of myeloid miRNAs, or contain several miRNAs in one transcrip- blasts in bone marrow and blood [1]. The long-term tional unit as miRNA clusters [3]. miRNAs post- overall survival (OS) rate for AML patients under the transcriptionally repress gene expression by recognizing age of 60 years and 60 years or older is 30–40% and complementary target sites in the 3 untranslated region under 10%, respectively [2], which remains a challenge. (UTR) of target mRNAs [4,5]. miRNAs have been impli- Thus, it is urgently needed to search for new targets for cated in a large variety of biological processes, including molecularly designed therapies. cell cycle progression, apoptosis, differentiation and haematopoiesis [6-10], and thereby play important roles in many pathological processes, including malignant * Correspondence: [email protected] transformation [11,12]. More than 50% of miRNA genes Equal contributors 1 are located in cancer-associated genomic regions or in Department of Hematology, Qilu Hospital, Shandong University, No.107, fragile sites, and both oncogenic and tumor suppressive Wenhua Xi Road, Jinan 250012 Shandong, P. R. China Full list of author information is available at the end of the article © Zhang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang et al. Molecular Cancer 2012, 11:56 Page 2 of 11 http://www.molecular-cancer.com/content/11/1/56 functions have thus far been ascribed to specific miR- treatment and after a complete remission and we found NAs [13]. Moreover, miRNAs have emerged as critical a lower miR-370 level at diagnosis while at least 2.1-fold regulators of hematopoiesis and their aberrant expres- increase in miR-370 expression after CR (Figure 1B). sion has been associated with the pathogenesis of leukemia [14]. Functional validation of deregulated miR- Changes in proliferation and cellular senescence of NAs in hematopoeisis has been shown for several miR- leukemic cells mediated by altered miR-370 expression NAs [15]. Distinctive patterns of increased expression We then explored the biological function of miR-370 in and/or silencing of multiple miRNAs have been asso- leukemic cells. Cells were transfected with precursors to ciated with specific cytogenetic and molecular subsets of miR-370 and miR-370 inhibitor to enhance and decrease AML [16]. miR-370 has been noted to be down- mature miR-370 expression, respectively. Transfection regulated in papillary thyroid carcinoma, colorectal can- with the miR-370 precursor increased mature miR-370 cer [17] and malignant cholangiocytes [18], but evidence expression 114.5 ± 5.70: 1 ± 0.12 (p < 0.05) and 59.8 ± of a biological role for this miRNA in AML has not been 6.90: 1 ± 0.24 (p < 0.05) (pSilencer-miR vs pSilencer) reported. In the present study, we sought to define the times higher in HL60 and K562 cells, respectively role of miR-370 in AML by investigating its expression (Figure 2A). Overexpression of miR-370 decreased cell and biological function in leukemic cell lines and blast proliferation (Figure 2C and Additional file 1) (pSilencer cells from patients with de novo AML. vs pSilencer-miR: HL60: 88 ± 15 vs 11 ± 4, p < 0.01; K562: 49 ± 5 vs 18 ± 5, p < 0.01). On the other hand, Results transfection with the miR-370 inhibitor suppressed mature Down-regulation of miR-370 expression in BM blasts from miR-370 expression to 31% ± 0.04 (p < 0.05) and 58% ± de novo AML patients 0.05 (p < 0.05) lower in HL60 and K562 cells, respectively We analyzed miR-370 expression in BM samples from (Figure 2B). The decline in miR-370 expression was 48 de novo AML patients at diagnosis time using qRT- coupled with enhanced cell proliferation (Figure 2C) (pSu- PCR. As shown in Figure 1A, the miR-370 level in per vs pSuper-miR-inhibitor: HL60: 56 ± 7 vs 72 ± 6, p < patients’ samples was significantly reduced (P < 0.01, 0.05; K562: 66 ± 12 vs 93 ± 7, p < 0.05). t test) compared to that from healthy controls, while fol- The above result suggests that miR-370 suppresses lowing acquisition of CR in the induction chemotherapy, proliferation of HL60 and K562 cells. We further wanted miR-370 expression level restored to 0.82 fold of con- to define the mechanism behind miR-370 overexpres- trols. There was no association between the presence of sion-mediated proliferation inhibition. We suspected mature miR-370 and age, gender, blast percentage or that miR-370 might trigger cellular senescence program. FAB subtypes (data not shown). In six patients, BM sam- Senescence-associated β-Gal staining, a specific marker ples were available both at diagnosis time prior to for senescent cells [19], was thus performed. A positive Figure 1 Downexpression of miR-370 in de novo AML patients. (A) miR-370 expression by qRT-PCR in 48 de novo AML patients, 40 AML st patients of 1 CR and 21 healthy controls. (B) Restoration of miR-370 expression in six patients after complete remission achievement in induction chemotherapy. The results are shown as miRNA expression after normalization with U6 and -ΔCt calculations. Zhang et al. Molecular Cancer 2012, 11:56 Page 3 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 2 (See legend on next page.) β-Gal staining was observed in the two cell lines trans- Up-regulation of miR-370 expression mediated fected with miR-370 precursors (Figure 2D) [pSilencer by 5-Aza-CdR vs pSilencer-miR (% of β-Gal-positive cells): HL60: 3 ± 1 DNA methylation is an epigenetic modification that reg- vs 28 ± 3, p < 0.01; K562: 8 ± 3 vs 40 ± 1, p < 0.01;]. ulates gene expression. Aberrant DNA methylation has Zhang et al. Molecular Cancer 2012, 11:56 Page 4 of 11 http://www.molecular-cancer.com/content/11/1/56 (See figure on previous page.) Figure 2 Suppression of AML cell proliferation and induction of cell senescence by overexpression of miR-370 in vitro while anti-miR-370 expression increases cellular ability of proliferation. (A) miR-370 expression in AML cell lines and its overexpression using a specific miR-expressing pSilencer vector. The cells were transfected with either miR-370 precursors or with control precursor and harvested after 48 hours. qRT-PCR was applied to detect mature miR-370 level. (B) The depletion of miR-370 expression by a specific miR-370-inhibiting pSuper plasmid. The method was similar as above. (C) Alterations in foci formation after different treatments in AML cells. The cells were first treated as above and the efficiently changed miR-370 expression was verified in (A) and (B). For clonogenic assays, 5,000 cells/well (in 6-well plates) were incubated for 14 days and the foci number was counted. (D) Senescence onset of miR-370-overexpressed AML cells. β-Gal staining was performed and % of positive cells was calculated. been implicated in many cancers [20]. Global hypo- (Figure 4C-D). There was a >2-fold increase in expres- methylation or aberrant hypermethylation of gene pro- sion of FoxM1 in HL60 and K562 cells after transfection moter CpG islands result, respectively, in tumor cell of miR370 inhibitor plasimids (Figure 4E). 5-aza-CdR genomic instability and gene silencing, particularly of significantly reduced the expression of FoxM1 in both tumor suppressor genes [17]. Interestingly, the chromo- HL60 and K562 cells (Figure 3 B-C). These changes somal location of miR-370 on chromosome 14q32.31 were similar to those observed with miR-370 overex- has been shown to be regulated by DNA methylation, or pression. Taken together, FoxM1 is a target of miR-370. deleted by loss of heterozygosity [14,21] or by hyper- methylation of an CpG island 200 bp upstream in the Overexpression of FoxM1 in de novo AML patients mother allele [22]. Treatment with 5 μM 5-aza-CdR, a FoxM1, a master regulator of mitotic gene expression, is DNA methylation inhibitor, for 72 hours, substantially required for cell proliferation and its inhibition leads to (>2.0-fold) and significantly (P < 0.05) increased the ex- reduction in anchor-independent growth and tumori- pression of miR-370 in both HL60 and K562 cells genesis of cancer cells [27]. As we have verified that (Figure 3A) and decreased cell proliferation (Figure 3D) FoxM1 is a target for miR-370, we then sought to probe (control vs CdR: HL60: 24 ± 4 vs 7 ± 2, p < 0.01; K562: its role in AML. The tumor specimens from forty-eight 152 ± 5 vs 78 ± 5, p < 0.001). de novo AML patients and forty AML patients in 1st CR and twenty-one healthy controls were analyzed for Identification of FoxM1 as a target for miR-370 FoxM1 mRNA expression using qRT-PCR. Patient char- To further elucidate the mechanism by which miR-370 acteristics are described in Table 1. The FoxM1 tran- affected cellular senescence and proliferation, we next script level in AML patients was found 21.47-fold higher screened for potential targets of miR-370 using four tar- than that in controls, while following acquisition of CR get prediction programs with different algorithms: in the induction chemotherapy, FoxM1 expression level DIANA-MicroT [23], TargetScan [24], Miranda [25] and reduced to 1.75 fold of controls. (Figure 5 A, C), which PicTar [26]. All potential targets predicted by more than was negatively correlated with miR-370 levels. There was one of these programs were identified. We selected the a highly significant difference in FoxM1 expression be- forkhead box M1 (FoxM1) for further study because of tween AML samples, CR samples and healthy controls its well-characterized role in tumor biology. The FoxM1 (qRT-PCR, One-Way ANOVA, p < 0.01). In six patients, gene has a 249-bp 3 UTR region that presents a 7-mer BM samples were available both at diagnosis time prior binding site for miR-370 (Figure 4A). to treatment and after a complete remission and we First, we made the luciferase reporter constructs con- found a higher FoxM1 level at diagnosis while a signifi- taining the miR-370 recognition sequence from the 3 - cant decrease in FoxM1 expression after CR except one UTR of FoxM1 inserted downstream of the luciferase sample (Figure 5B). There was no clear association be- gene. Transfection with miR-370 precursor decreased re- tween the presence of FoxM1 mRNA and age, gender, porter activity in K562 cells (Figure 4B), which strongly tumor burden or FAB subtypes (data not shown). More- indicates that FoxM1 is a target for miR-370. Next, the over, BM materials from 8 de novo AML patients, 8 studies were repeated with random mutations in the rec- patients in 1st CR and 5 healthy controls randomly ognition sequence (Figure 4A), which resulted in aboli- chosen from our AML patient pool were used to deter- tion of the reporter activation by miR-370 precursor mine the mRNA expression level of c-Myc, hTERT, kip1 (Figure 4B). Finally, we assessed the effect of miR-370 p27 and skp2, all of which were the target genes of expression on FoxM1 expression. Transfection of HL60 FoxM1, using real-time PCR method. We found that the and K562 cells with miR-370 precursor resulted in lower transcript levels of c-Myc, hTERT and skp2 in AML expression of FoxM1 after 48 hours (Figure 4C-D). Con- patients were respectively 9.64, 3.76 and 3.14-fold higher comitant with decreased FoxM1 expression, there was than those in controls, while following acquisition of reduction of its downstream target c-myc and skp2 CR, all of them reduced almost to the same levels of Zhang et al. Molecular Cancer 2012, 11:56 Page 5 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 3 Expression of miR-370 in AML cells (HL60 and K562) and the effect of 5-aza-2’deoxycytidine (5-Aza-CdR). AML cells were plated in 6-well plates, treated with 5 μM 5-Aza-CdR or PBS and harvested after 72 hours. The expression of mature miR-370 was assessed using real-time PCR. (A) 5-aza-CdR increases the relative expression of miR-370 in AML cells. Data represent mean ± s.e. from three separate determinations. *p < 0.05 compared with respective controls. (B) 5-aza-CdR decreases the relative mRNA level of FoxM1 in AML cells. *p < 0.05. (C) 5-aza-CdR decreases the protein level of FoxM1 in AML cells. (D) Diminished foci formation of 5-aza-CdR treated AML cells. kip1 controls. On the contrary, the expression level of p27 regulated and coupled with the accumulation of the kip1 in AML patients is only 40% of that in controls, while CDK inhibitor p27 (Figure 5 D-E). restored after CR (Additional file 2). Collectively, the FoxM1 gene is aberrantly activated in AML and is required for sustained proliferation of the Defective foci formation by depletion of FoxM1 in AML cancer cells. cell lines Then, we employed the FoxM1-specific siRNA to knock Discussion down FoxM1 expression in AML cell lines and the cells In the present study, we explored the expression and were then assessed for their clonogenic capacity. Effi- role of miR-370 in AML. Our findings show a down- cient silence of FoxM1 expression in these cells was regulation of miR-370 in blasts from patients with de verified using qRT-PCR and Western blot analyses novo AML. Moreover, we identified FoxM1 as a target (Figure 5 D-E). Compared to the control cells, the for miR-370 and restored expression of miR-370 reduced FoxM1-knocked down cells exhibited significantly the level of FoxM1. In hematological malignancies a link diminished foci formation (Figure 5F) (Controls vs with miRNA was initially described in chronic lympho- FoxM1 siRNA: HL60: 19 ± 3 vs 11 ± 2, p < 0.05; K562: cytic leukemia (CLL) by Calin et al. [28]. A cluster of 33 ± 5 vs 5 ± 2, p = 0.001). Following FoxM1 depletion, two miRNAs, miR-15a, and miR-16, was found to be its target genes c-myc, skp2 and hTERT were also down- located within the deleted region at 13q14, and down- Zhang et al. Molecular Cancer 2012, 11:56 Page 6 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 4 Identification of FoxM1 as a target of miR-370. (A) Predicted site of miR-370 in the FoxM1 3’ UTR mRNA. (B) Luciferase activities were determined at 24 hours and were normalized by Renilla luciferase activity. K562 cells were plated in 24-well plates, and then transfected with 0.05 μg of a Renilla luciferase expression construct pRL-TK and 0.5 μg of the pGL3-FoxM1-wt-luc or pGL3-FoxM1-mut-luc firefly luciferase expression construct, along with either miR-370 precursor or control precursor. Luciferase assays were performed after 24 hours using the dual luciferase reporter assay system. The expression of firefly luciferase activity was normalized to that of renilla luciferase activity for each sample. A decrease in relative firefly luciferase activity in the presence of miR-370 indicates the presence of a miR-370 modulated target sequence in the 3’- UTR of FoxM1. Data represents mean ± s.e. from three separate experiments. *P < 0.05. (C) and (D) The expression of FoxM1 and downstream genes were regulated by miR-370 overexpression at both transcription level and translation level. Western blots were performed and sequentially kip1 probed with antibodies against FoxM1, c-Myc and p27 , β-actin was a loading control. Efficient miR-370 overexpression was verified in Figure 2 (A). (E) Increase in FoxM1 protein level after anti-miR-370 treatment. Efficient miR-370 depletion was verified in Figure 2 (B). regulated in the majority (70%) of CLL samples. Notably, with ALL on the basis of 21 up-regulated and 6 down- miR-15a and miR-16-1 down-regulation contribute to regulated miRNAs, among which four of them (let-7b, malignant transformation by up-regulating BCL2 [29]. miR-128a, miR-128b and miR-223) were the most dis- Recently, involvement of miRNA in AML has been criminatory [31]. According to Amanda Dixon-McIver, documented identifying that miRNA expression profiles compared with other major cytogenetic subgroups of are AML subtype-specific and suggesting a pathogenetic AML, APLs bearing the t(15;17) translocation were role of miRNA in AML. For example, Mi and colleagues characterised by the up regulation of 7 miRNAs tran- [30] showed distinct miRNA signatures between acute scribed from genes located at the 14q32 region. The set lymphoblastic leukemias (ALLs) and AMLs. In that includes miR-127, miR-154, miR-154*, miR-299, miR- study, patients with AML could be separated from those 323, miR-368, and miR-370. Two other groups reported Zhang et al. Molecular Cancer 2012, 11:56 Page 7 of 11 http://www.molecular-cancer.com/content/11/1/56 Table 1 Patient Characteristics cancer development [35]. For instance, miR-127 has Characteristic De novo AML patients been shown to be down regulated or silenced in cancer st AML patients in 1 CR cells, whose expression is correlated with the methyla- Patients,no. 48 40 tion and acetylation status of its promoter. Inhibition of methylation and histone deacetylation in these cancer Sex,male/female,no.(%) 31(65)/17(35) 24(60)/16(40) cells causes over expression of miR-127 and related Median age,y(range) 41(14–74) 41(14–68) down regulation of the target BCL6, a bona fide pro- WBC count (10 /L) tooncogene [36]. We hypothesize that miR-370 also Median 46.3 6.3 acts as a tumor suppressor in AML, as in papillary Range 0.8-354.9 2.6-10.2 thyroid carcinoma, colorectal cancer and malignant Hemoglobin (g/dL) cholangiocytes. The comparison of the leukemia sam- ples with healthy controls highlighted the differential Median 7.7 11.8 expression of miR-370. Following the treatment with Range 4.1-13.2 8.3-16.1 5-aza-2 -deoxycytidine, there is a significant enrich- Platelet count (10 /L) ment for miR-370 in AML cell lines, which indicated Median 58 243.3 that hypermethylation may contribute to reduction of Range 2-319 76-384 miR-370. BM blasts (%) Cancer therapy has traditionally relied on cytotoxic treatment strategies on the assumption that complete Median 69.1 2.1 cellular destruction of tumors optimizes the potential for Range (24–96) (0–5) patient survival. Although these approaches produce FAB subtype,n/(%) complete cell death within a tumor, they also can cause M2 9/(19) 6/(15) severe side effects in patients [37]. Recently, a promising M3 11/(23) 20/(50) approach to preventing continued tumor growth is M4 11/(23) 8/(20) therapy-induced senescence (TIS) [38]. Senescent cells remain viable and metabolically active but are perman- M5 15/(31) 5/(12.5) ently growth arrested [39]. Evidence has recently accu- M6 2/(4) 1/(2.5) mulated that cellular senescence is a potent barrier to cancer development. Our results indeed demonstrated independently that miR-155 was up-regulated in AML that senescence occurred in most of AML cells treated patients with FLT3-ITD, suggesting that this miRNA with miR-370 overexpressing plasmid, which was con- contributes to the highly proliferative phenotype of this comitant with their diminished clonogenic capacity. molecular subset of AML [31-33]. In the present study, Given a critical role for senescence induction in tumor we haven’t got the conclusion that the expression level suppression and therapeutic efficacy of cancer treat- of miR-370 is with AML subtype-specificity, which may ment, the present findings have important biological be due to the limited number of primary AML patients and clinical implications. All these results suggest that enrolled in our study. downregulation of miR-370 may be another mechanism DNA methylation is an epigenetic modification that involved in the pathology of AML and therefore, could can regulate gene expression. Aberrant DNA methyla- be used as a diagnostic marker and therapeutic target tion has been implicated in many cancers [21]. miR-370 in AML. and many other miRNAs are organised in clusters to- We have also analyzed the correlation between miR- gether on chromosome 14q32 [34]. This miRNA cluster 370 expression and FoxM1 mRNA expression in 48 de acts as imprinted non-coding RNA (ncRNA) genes, novo AML samples. Consistent with the cell line data, which are mono-allelically expressed in a parent-of- FoxM1 was enriched in the primary blasts mRNAs that origin manner (their expression is restricted to either inversely correlated to miR-370 expression levels. This the maternal or the paternal allele). Interestingly, as for result was also observed at the protein level in a few pri- this miRNA cluster, which is of maternal origin, its mary AML samples. However, further studies using large imprinted expression is regulated by an intergenic differ- numbers of primary AML samples will be needed to entially methylated region (DMR) located ~ 200 kb up- confirm this interaction. stream from the miRNA cluster [24]. Hypermethylation of DMR causes silence of this miRNA cluster, including Conclusion miR-370. It has been suggested that miRNAs in this re- We demonstrate that miR-370 is a tumor suppressive gion act as tumour repressor genes and that changes in factor by targeting multiple critical oncogenic pathways. the methylation status of their promoters could trigger Restoring miR-370 expression downmodulates FoxM1, Zhang et al. Molecular Cancer 2012, 11:56 Page 8 of 11 http://www.molecular-cancer.com/content/11/1/56 Figure 5 Overexpression of FoxM1 in de novo AML patients and defective foci formation induced by depletion of FoxM1 in AML cell lines. (A) and (C) Expression of FoxM1 mRNA and protein in de novo AML patients, AML patients of 1st CR and controls, were determined using qRT-PCR and Western blot respectively. (B) Decrease of FoxM1 expression in six patients after complete remission achievement in induction chemotherapy. (D) and (E) FoxM1 depletion triggers concomitant down-regulation of c-myc, hTERT and skp2 coupled with the accumulation of kip1 the CDK inhibitor p27 in AML cells. The cells were transfected with either control or FoxM1 siRNA and harvested after 72 hours. qRT-PCR and western blot were applied to detect mRNA and protein levels, respectively. (F) Diminished foci formation of FoxM1-depleted AML cells. The cells were first treated as above and the efficient FoxM1 knock down was verified in (D) and (E). induces senescence, and dampens cell growth in AML presentation and morphologic criteria of the French- cells, thereby suggesting miRNA-based therapy as a American-British (FAB) Classification. The study was novel approach to increase response in AML. approved by the local ethics committee. Patients’ BM samples were collected between April 2008 and Septem- Materials and methods ber 2011 at the Department of Hematology, Qilu Hos- Patients and bone marrow samples pital, Shandong University, Jinan, China. Mononuclear Forty-eight newly diagnosed AML patients (31 male and cells were isolated using Ficoll-Hypaque density gradient 17 female; median age at diagnosis 41; range 14–74), centrifugation, and then stored at −80°C until use. All st forty AML patients in 1 complete remission (CR) and patients and healthy controls were tested for miR-370 twenty-one healthy controls were enrolled in this study. and FoxM1 mRNA levels in their BM cells. Among Diagnosis of AML was established according to clinical those AML patients, six were analyzed for miR-370 and Zhang et al. Molecular Cancer 2012, 11:56 Page 9 of 11 http://www.molecular-cancer.com/content/11/1/56 FoxM1 levels in their bone marrow samples at both Western blot diagnosis and complete remission. Total cellular proteins were extracted from cultured cells or BM samples. Proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The mem- Cell lines and culture conditions branes were probed with the specific antibodies against Human AML cell lines HL60 and K562 were cultured at FoxM1, p27, c-MYC (Santa Cruz Biotechnologies, Santa 37°C, 95% air, 5% CO in RPMI 1640 containing 10% Cruz, CA, USA), followed by anti-mouse or rabbit horse- heat-inactivated fetal bovine serum (FBS; Gibco, radish peroxidase–conjugated IgG and developed with Carlsbad, CA, USA), 100 μg/mL penicillin, and 50 μg/mL the enhanced chemiluminescence method (ECL). β-actin streptomycin. To assess 5-aza-CdR (Sigma, Santa Clara, served as a loading control. CA, USA) effects, cells were grown on 6-well plates, trea- ted with 5 μM 5-aza-CdR or cold phosphate-buffered sa- miR-expressing and miR-inhibiting plasmids line (PBS) controls for 72 h at 37°C, and then harvested To generate the miR-expressing pSilencer3.1-H1 neo for isolation of total mRNA or protein. vector, a fragment of 212 base pairs (bp) corresponding to the desired miRNA and the surrounding sequences RNA extraction and quantitative real-time PCR was amplified from human genomic sequence, adding a 0 0 Total cellular RNA in BM samples and in cells with dif- BamHI site and a HindIII site to the 5 and 3 ends re- ferent treatments was extracted using the Trizol (Invitro- spectively, using the polymerase chain reaction (PCR) gen, Carlsbad, CA, USA) according to the manufacturer’s with primers sense: 5 AAGGGATCCTACTTGAGG 0 0 protocol. cDNA was synthesized using random primers GATGGGCGATA 3 and antisense 5 TCAAAGCTTC (N6) (Fermentas, St. Leon-Rot, Germany) and MMLV re- CCGAGCTCTGGTGTTAGAC 3 . We included a large verse transcriptase. The PCR primers used in the study portion of miRNA surrounding sequence in the attempt were as follows: sequences specific for FoxM1 mRNA: 5 - to allow correct processing of the miRNA to its mature 0 0 TGCAGCTAGGATGTGAATCTTC-3 (Forward) and 5 - form and to induce overexpression while preserving a GGAGCCCAGTCCATCAGAACT-3 (Reverse). Skp2: physiologic mechanism of miRNA production. miR-370 0 0 5 -GGACCTATCGAACTCAGTTAT-3 (Forward) and inhibitor sequences were synthesized as DNA oligonu- 0 0 kip 5 -CAGCCACCTGTACATGCTTT-3 (Reverse); p27 : cleotides; after annealing, were sticking ended and sub- 0 0 5 -ATGTCAAACGTGCGAGTGTCTAA-3 (Forward) and cloned into a pSuper vector. 0 0 5 -TTACGTTTGACGTCTTCTGAGG-3 (Reverse); hTERT: 0 0 0 5 -CGGAAGAGTGTCTGGAGCAA-3 (Forward) and 5 - Transfection 0 0 GGATGAAGCGGAGTCTGGA-3 (Reverse); c-MYC: 5 - Cells were incubated in 6-well plates (3.0× 10 /well and TACCCTCTCAACGACAGCAGCTCGCCCAACTCCT-3 1.0× 10 /well, respectively) overnight and were then (Forward) and 5 -TCTTGACATTCTCCTCGGTGTCC transfected with plasmid or siRNA using Lipofectamine GAGGACCT-3 (Reverse). The above primer pairs cross 2000 (Invitrogen, Carlsbad, CA, USA) according to the intron/exon boundaries; thus, the resultant PCR products manufacturer’s protocol. Chemical modified Stealth small do not represent genomic DNA contamination. β-actin interfering RNA (siRNA) targeting FoxM1 and control expression was used as a control for RNA loading and RT siRNA were bought from Invitrogen. The sequence for efficiency and amplified. Quantitative real-time polymerase the FoxM1 siRNA was 5 -GACAACUGUCAAGUGUAC chain reaction (qRT-PCR) was carried out in an ABI7000 CACUCUU-3 . sequence detector (Applied Biosystems, Foster City, CA, USA). Soft agar colony formation assay HL60 and K562 cells were resuspended in DMEM (Gibco, TaqMan qRT-PCR miRNA analysis Carlsbad, CA, USA) containing 20% heat-inactivated fetal Quantification of mature miRNAs was performed using bovine serum (FBS; Gibco, Carlsbad, CA, USA) with equal qRT-PCR with the TaqMan miRNA assay kit (Applied amount of either 0.3% agar (HL60 cells) or 0.5% agar Biosystems, Foster City, CA, USA) according to manufac- (K562 cells), and plated in 6-well plates at 5,000 per well turer’s instruction. Briefly, 10 ng of total RNA was re- on top of a 2 mL precast semisolid 1% agar underlayer as verse-transcribed (RT) with specific primers, subsequently described previously [40]. The number of colonies with 1.5 μL of RT product was used as template for real-time more than 50 cells was counted after two weeks. PCR. All real-time experiments were performed in tripli- cate. Data were normalized by the expression of small Senescence-associated β-Galactosidase nuclear RNA (snRNA) U6 and expressed either as rela- (SA -β-Gal) staining -ΔCt tive expression (2 ) or as fold change relative to con- SA -β-Gal staining was done as described [19,41]. Briefly, -ΔΔCt trol (2 ). the cells grown in 6-well plates were transfected with Zhang et al. Molecular Cancer 2012, 11:56 Page 10 of 11 http://www.molecular-cancer.com/content/11/1/56 pSilencer or pSilencer-miR-370. After 7 days, the cells were Acknowledgements This work was supported by the National Natural Science Foundation of rinsed with PBS once, fixed in 3% of formaldehyde for China (Nos. 81170514, 30971151, 81172354, 30972775 and 81001098), the 15 min, and incubated with freshly prepared SA -β-Gal Project Sponsored by the Scientific Research Foundation for the Returned staining solution at 37°C overnight. Overseas Chinese Scholars, State Education Ministry (The Project-sponsored by SRF for ROCS, SEM), Shandong Provincial Natural Science Foundation, China (Nos. ZR2009CM002, ZR2009CZ001, ZR2010HZ003 and ZR2012HQ003), Luciferase reporter vector Independent Innovation Foundation of Shandong University (IIFSDU, Nos. 2010TS074 and 2012TS106) and the National Basic Research Program of The precursor to miR-370 was synthesized and cloned in China (973 Program, 2012CB911202). pSilencer. Firefly luciferase reporter vectors with the intact putative miR-370 recognition sequence from the 3 -UTR Author details Department of Hematology, Qilu Hospital, Shandong University, No.107, of FoxM1 (pGL3-FoxM1-wt-3 -UTR) or with random 0 Wenhua Xi Road, Jinan 250012 Shandong, P. R. China. Department of mutations (pGL3-FoxM1-mut-3 -UTR) cloned down- 3 Biochemistry, Shandong University, Jinan, China. Department of stream of the firefly luciferase gene were constructed. Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, Jinan, China. Wild-type and mutant inserts were confirmed by sequen- 0 Department of Medicine, Division of Hematology and CMM, Karolinska cing. 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Published: Aug 17, 2012

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