MicroRNA let-7a-3 gene methylation is associated with karyotyping, CEBPA promoter methylation, and survival in acute myeloid leukemia

Leukemia Research 38 (2014) 625–631

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MicroRNA let-7a-3 gene methylation is associated with karyotyping, CEBPA promoter methylation, and survival in acute myeloid leukemia Ya-Chen Ko a , Woei-Horng Fang b,c , Tsung-Chin Lin b , Hsin-An Hou d , Chien-Yuan Chen d , Hwei-Fang Tien d , Liang-In Lin b,c,∗ a

Department of Medical Imaging and Radiological Technology, Yuanpei University, Hsinchu, Taiwan Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan c Department of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan d Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan b

a r t i c l e

i n f o

Article history: Received 25 November 2013 Received in revised form 5 March 2014 Accepted 6 March 2014 Available online 19 March 2014 Keywords: Acute myeloid leukemia miRNA let-7a-3 CCAAT/enhancer binding protein ␣ DNA methylation

a b s t r a c t Let-7a-3 transcribes the miRNA let-7a, of which the expression is dysregulated in cancer. We evaluated the significance of let-7a-3 gene methylation in patients with de novo acute myeloid leukemia (AML). Let-7a-3 was methylated in 81.1% (73/90), partially methylated in 12.2% (11/90), or unmethylated in 6.7% (6/90) of patients. Let-7a-3 methylation correlated with AML karyotyping and CCAAT/enhancer binding protein ␣ (CEBPA) methylation. Kaplan–Meier survival analysis predicted that let-7a-3 hypermethylation correlated with better survival in AML with hypomethylated CEBPA or with hypomethylated CEBPA without the favorable karyotype. We conclude that let-7a-3 methylation is a positive prognosticator for AML patients with hypomethylated CEBPA. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Acute myeloid leukemia (AML) is an aggressive malignant disorder of hematopoietic cells resulting from a number of heterogeneous genetic abnormalities. For example, chromosomal translocations, including t(15;17), t(8;21), and inv(16), or a normal karyotype with mutations in FLT3-ITD, nucleophosmin (NPM1), or CCAAT/enhancer binding protein ␣ (CEBPA) genes, may underlie AML. AML patients are classified into favorable, intermediate, or unfavorable cytogenetic risk groups [1–3]. An AML patient’s genetic background determines disease prognosis. Several molecular mutations have recently been identified as AML prognosticators [4], including AML1/RUNX1, WT1, and DNMT3A mutations as well as CEBPA methylation [5–8]. Furthermore, specific expression patterns of certain microRNA (miRNA) molecules are associated with karyotypes, bone-marrow histopathology, molecular markers, or clinical outcomes in AML [9].miRNAs are endogenous small noncoding RNAs of approximately 22 nucleotides. They repress gene expression post-transcriptionally by targeting the 3 untranslated

∗ Corresponding author at: Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, No. 1, Chang-Te St., Taipei, Taiwan. Tel.: +886 2 23123456x67342; fax: +886 2 23711574. E-mail address: [email protected] (L.-I. Lin). http://dx.doi.org/10.1016/j.leukres.2014.03.008 0145-2126/© 2014 Elsevier Ltd. All rights reserved.

regions of certain mRNAs, mediating mRNA cleavage or translation inhibition [10,11]. miRNAs are potential therapeutic agents or targets [12,13] because dysregulated miRNA expression has been described in multiple malignancies. The human miRNA let7a of the let-7 family originates mainly from the let-7a-3 gene. The human let-7a sequence is identical to that of Caenorhabditis elegans, suggesting conserved functions. Let-7 was initially described to control cell proliferation and differentiation during C. elegans larval development [14]. Subsequent studies showed that let-7 family members target the HMGA2 oncogene and some cell-cycle regulators, including CDC25A, CDK6, E2F2, and CCND2 [15–17]. Let-7a miRNA is believed to suppress tumorigenesis by targeting Ras and Myc oncogenes [18,19]. Furthermore, its expression was found to be low in head-and-neck cancers, prostate cancer, or melanomas [17,20,21]. In contrast, let-7a dysregulation may contribute differentially to tumorigenesis in various cell types; for example, let-7a expression is up-regulated in diffuse large B-cell lymphoma [22]. Epigenetically, gene expression is controlled by methylation of cytosine residues located in CpG-rich regions. Methylation in the promoter CpG regions often causes gene silencing by affecting chromatin condensation and inhibition of transcription-factor binding. Gene-promoter CpG islands are less methylated in normal cells. In cancer cells, however, promoter regions are usually hypermethylated, causing gene silencing. Altered epigenetic control of gene expression plays an important role in oncogenesis, including

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myeloid leukemogenesis. In AML, hypermethylation is observed in a number of genes involved in leukemogenesis. These include p15, E-cadherin, SOCS-1, p73, DAPK1, HIC1, RARˇ2, and CEBPA [8,23–25]. Epigenetic control is also important in regulating miRNA functions. Human let-7a-3 gene is located in a CpG island where its expression is regulated epigenetically [26]. However, let-7a-3 is differentially methylated in tumor tissues and cancer cell lines [26–28]. Additionally, methylation of let-7a-3 is linked to favorable prognoses in ovarian cancer [27]. Therefore, studying regulation and functions of miRNAs involved in myeloid leukemogenesis is important for advancing therapeutic and prognostic measures. Here, we examined the methylation status of let-7a-3 in AML cell lines and 90 AML patient samples. Correlations between let-7a3 methylation and clinical features and AML prognosis were also assessed. 2. Materials and methods 2.1. Patients and study samples From 2001 to 2004, 90 patients diagnosed with de novo AML in the National Taiwan University Hospital (NTUH) were enrolled in this study. Informed written consent was obtained from the patients in accordance with the Declaration of Helsinki, and the study was approved by the Institutional Review Board of the NTUH. These studied patients were aged from 17 to 90 (median: 47) and designated P1 to P90. AML diagnosis and classification were established according to the French–American–British (FAB) Cooperative Group Criteria. All patient samples were obtained at disease onset. Bone-marrow-derived mononuclear cells (BM-MNCs) were collected by Ficoll–Hypaque density gradient. Leukemic cells comprised over 90% of cell population.

2.4.2. Combined bisulphite—Restriction analysis (COBRA) Nested-PCR reactions on the bisulphite-treated genomic DNA were performed as previously described by Brueckner et al. [26] to generate the 723-bp product. This product was then gel-purified, cut with BstUI and electrophoresed on 3% agarose gels. Samples were recognized as let-7a-3 methylated (M) when more than 70% of the 723-bp fragment were digested by BstUI as revealed on the gels; samples were recognized as unmethylated (U) when less than 30% of the 723-bp fragment was digested; whereas other samples were recognized as partially methylated (P). 2.4.3. Bisulphite sequencing The 723-bp PCR products were cloned using the pGEM-T Easy Vector System (Promega, Madison, MI, USA) as per Promega instructions. Methylation patterns were determined up to 10 clones each using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and an automated ABI 3730 Genetic Analyzer (Applied Biosystems) as stated previously [8]. 2.5. Cytogenetic study Chromosome analysis was performed as described previously [29]. Briefly, bone-marrow cells from patients were used immediately or after 1–3 days of nonstimulated culturing. Metaphase chromosomes were visualized by the conventional trypsin–Giemsa banding technique and were karyotyped according to the International System of Human Cytogenetic Nomenclature. 2.6. Mutational analysis and CEBPA methylation Mutational analyses in the genes of CEBPA, AML1, KIT, PTPN11, WT1, N-RAS, K-RAS, NPM1, FLT3-ITD, FLT3-TKD and MLL-PTD were performed as described previously [5,30–33]. CEBPA methylation status in the distal promoter region was evaluated by quantitative MassARRAY analyses using Sequenom® Standard EpiPanel (Sequenom, San Diego, CA, USA) as described previously [8,34]. Accordingly, CEBPA methylation levels were calculated as the mean methylation values of three CpG units (units 2, 3, and 4) covering seven CpG sites; and with cutoff methylation level of 0.486, the AML patients were categorized into two groups with hypermethylated and hypomethylated CEBPA.

2.2. Cell lines 2.7. Statistics A549 cells were cultured in Dulbecco’s Modified Eagle Media and leukemia cell lines, HEL, HL60, and U937 were cultured in RPMI 1640, both supplemented with 10% fetal bovine serum at 37 ◦ C in 5% CO2 . 2.3. RNA extraction, reverse transcription, and let-7a miRNA quantification Cellular RNA from patient samples or cell lines were purified using RNAzolTM B Reagent (Tel-Test, Friendswood, TX, USA) and reverse-transcribed into cDNA using the let-7a multiplex stem–loop reverse primer, and the TaqMan miRNA Reverse Transcription kit (Applied Biosystems). Following cDNA synthesis, let-7a miRNA was quantified by real-time quantitative polymerase chain reaction (PCR) using TaqMan MicroRNA Assay (Applied Biosystems) as per manufacturer’s instructions. An ABI PRISM 7300 detection system (Applied Biosystems) was used for quantitative PCR. Initial denaturation at 95 ◦ C for 10 min was followed by 40 thermal cycles comprising 95 ◦ C for 15 s and 60 ◦ C for 1 min. RNU48 expression was analyzed as the internal PCR control. The relative let-7a to RNU48 expression was calculated as 2−CT where CT = CT(let-7a) − CT(RNU48) . 2.4. Bisulphite reaction and DNA methylation analysis 2.4.1. Bisulphite treatment of genomic DNA Bisulphite treatment of genomic DNA was performed using the EZ DNA Methylation-Gold kit (ZYMO Research, Irvine, CA, USA) as per manufacturer’s instructions.

Chi-squared tests or Fisher’s exact tests were performed to calculate correlations between let-7a-3 COBRA results and patients’ age, gender, FAB subtype, karyotyping, gene mutations and methylation of the distal CEBPA promoter. Survival of patients with hypermethylated and hypomethylated let-7a-3 was estimated by Kaplan–Meier analyses and log–rank tests. A p value of <0.05 indicated statistical significance. All statistical analyses were performed with SPSS Version 18 software and Statsdirect (2.7.8b, 2011).

3. Results 3.1. Let-7a-3 gene was heavily methylated in AML cells Let-7a-3 methylation was detected in HEL, HL60, K562, and U937 leukemia cells by using COBRA analysis, which examines the let-7a3 methylation status at the 20 21 and 26 27 CpG sites (2 BstUI sites, Fig. 1 and Supplementary Fig. S1a). We then assessed methylation levels of 33 CpG sites using bisulphite sequencing, and demonstrated that 93.3%, 81.2%, 70.7%, and 89.7% of the CpG sites were methylated in HEL, HL60, K562, and U937 cells, respectively (Fig. 2). These results showed that let-7a-3 was heavily methylated in these 4 AML cell lines.

Fig. 1. Assessment of let-7a-3 methylation. The human let-7a-3 gene is embedded in a CpG island on chromosome 22. Methylation at 33 CpG sites in let-7a-3 gene was examined by nested PCR generating a 723-bp PCR fragment containing the primary let-7a-3 miRNA sequence (Black Box). The genomic location of the fragments shown in this figure correspond to the GeneBank accession number, NT 011520 (from nt25898610 to nt25899401). The vertical lines indicate locations of the 33 CpG sites within this region. Two BstUI restriction sites included in this region (CpGs 20 21 and CpGs 26 27) are indicated by vertical arrows. Open and filled horizontal arrows indicate locations of the first and second sets of primers for nested PCR, respectively. For correlation analyses, the untranscribed regions in the gene fragment were divided into segments 1 through 7 as indicated.

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Fig. 2. Methylation level of let-7a-3 gene assessed by bisulphite sequencing. (a) Bisulphite sequencing results in AML cell lines and 4 AML patients. : unmethylated CpG dinucleotide; 䊉: methylated CpG dinucleotide. (b) Methylation percentages at 33 CpG sites of let-7a-3 gene in AML cell lines and 16 AML patients. Total methylation percentage is calculated from bisulphite-sequencing results of up to 10 clones from each sample. M, methylated; P, partially methylated; U, unmethylated.

3.2. Let-7a-3 gene was heavily methylated in most patients with AML By using COBRA to assess BM-MNCs harvested from the 90 AML patients, let-7a-3 was found to be methylated in 81.1% (73/90), partially methylated in 12.2% (11/90), or unmethylated in 6.7% (6/90) of patients (Supplementary Fig. S1b). Bisulphite sequencing results from three unmethylated, five partially methylated, and eight methylated randomly chosen samples revealed the extent of

methylation ranging from 11.8% to 14.8%, 43.3% to 63.6%, and 56.4% to 86%, respectively (Fig. 2b), demonstrating bisulphite sequencing results correlated highly with the COBRA data. In addition, most of the AML patients harbored a heavily methylated let-7a-3 gene. Although methylation at 4 CpG sites (Fig. 1; numbered 20, 21, 26, and 27) assessed by COBRA method showed a high correlation with the extent of methylation at 33 CpG sites assessed by bisulphite sequencing, we wonder whether a minimum methylation sites could represent methylation status of 33 CpG sites more.

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Table 1 Extent of methylation (%) at CpG sites in let-7a-3 gene. Samples

HEL HL60 K562 U937 P4 P19 P25 P26 P28 P33 P48 P50 P51 P52 P53 P56 P67 P76 P77 P86 Rb

33 CpG sitesa

93.3 81.2 70.7 89.7 13.3 11.8 43.3 85.5 71.5 78.5 82.6 79.4 84.2 86.0 44.8 63.6 44.5 57.6 56.4 14.8

Segment 1

2

3

4

5

6

7

97.5 80.0 69.4 95.0 0 0 20.0 67.5 80.0 83.3 93.5 37.5 86.6 81.2 35.0 42.5 42.5 16.6 12.5 15.0 0.874

100 88.3 74.0 88.3 3.3 34.2 16.6 83.3 83.3 81.4 81.2 78.3 92.5 93.7 55.0 77.9 31.6 42.5 43.3 13.3 0.925

96.6 63.3 37.0 60.0 6.6 0 16.6 93.3 56.6 59.2 33.3 73.3 55.5 79.1 13.3 50.0 26.6 5.5 33.3 6.6 0.855

46.6 50.0 59.2 90.0 0 0 40.0 5.3 23.3 51.8 62.5 56.6 55.5 56.5 13.3 36.6 23.3 13.3 40.0 13.3 0.722

95.0 84.2 86.1 92.5 0 2.5 50.0 95.0 70.0 81.9 89.0 96.2 90.2 77.0 42.5 53.7 45.0 87.5 60.0 12.5 0.954

100 90.0 72.2 93.3 21.6 1.6 66.6 91.6 73.3 87.0 93.7 94.9 90.7 95.8 51.6 80.0 50.0 61.1 91.6 13.3 0.950

100 80.0 66.6 100 90.0 76.6 93.3 100 100 85.1 100 90.0 96.2 100 93.3 96.6 100 100 100 36.6 0.490

Segments 1–7 were represented in Fig. 1. a Percentages were calculated using bisulphite-sequencing results of 10 clones from each sample. b Correlation coefficient.

Within this PCR-amplified 723-bp fragment of let-7a-3 gene, we divided it into segments 1 through 7 from upstream to downstream (Fig. 1). The numbers of methylation sites were 4, 6, 3, 3, 8, 6, and 3 for segments 1 to 7, respectively. Correlation analyses showed that methylation levels at segments 1 through 6 each independently correlated highly with the overall percentage of let7a-3 methylation (R = 0.874, 0.925, 0.855, 0.722, 0.954, and 0.950, respectively; Table 1), in which the highest correlations were found in segments 5 and 6, but segment 7 showed only a moderate correlation (R = 0.490). In this study, the 2 BstUI restriction sites which COBRA method examined are located in segments 5 and 6. High correlation of methylation percentage of fragments 5 and 6 with overall percentage of let-7a-3 methylation was noted (R = 0.954, and 0.950; Table 1). 3.3. Let-7a-3 methylation was inversely correlated with let-7a miRNA expression It was previously demonstrated that let-7a-3 expression is epigenetically controlled by promoter methylation [26,27,35]. To assess correlations between let-7a-3 expression and methylation, let-7a miRNA were quantified in 3 cell lines and 6 available AML RNA samples (Supplementary Fig. S2). As expected, P67 with partially methylated let-7a-3 expressed higher levels of let-7a than other patients (P55, P61, P72, P74, and P88) or cell lines with methylated let-7a-3, showing a tendency of an inverse correlation between let-7a-3 methylation and let-7a expression. 3.4. Let-7a-3 methylation was associated with karyotyping and CEBPA methylation in AML According to let-7a-3 status, 87 AML patients with available clinical data were categorized into three groups with unmethylated, partially methylated and methylated let-7a-3. Let-7a-3 methylation was correlated with patients’ karyotypes, but not with age, gender, FAB subtypes (Table 2), and several AML-associated gene mutations (Supplementary Table 1). The group with favorable karyotypes [t(15;17), t(8;21), and inv(16)] harbored methylated let-7a-3

less frequently (25%) than those with intermediate [normal and others; 80.3% (49/61)] or unfavorable [(−5, −7, +8 and complex; 94.4% (17/18)] karyotypes (p = 0.002) (Table 2). Methylation status of let-7a-3 correlated inversely with methylation status of the distal promoter region of CEBPA in AML patients (p = 0.042) (Table 2). We also analyzed let-7a-3 methylation levels in AML patients with different genetic alterations and observed that all patients carrying CEBPA mutations harbor methylated let-7a-3 (12/12, 100%; Supplementary Table 1), although correlation between let-7a-3 methylation and CEBPA mutations is not statistically significant (p = 0.196). Furthermore, we demonstrated that let-7a-3 methylation is associated with the presence of FLT3-TKD mutations (Supplementary Table 1), in which patients with FLT3-TKD mutations harbor methylated let-7a-3 less frequently (3/8; 37.5%) than those without the mutations (65/79; 82.3%; p = 0.019).

3.5. Let-7a-3 methylation was associated with AML patients’ survival Of the 60 patients receiving standard induction therapy, 45 (75.0%) patients achieved a complete remission (CR). For prognosis analyses, these 60 AML patients were divided into 2 groups with hypermethylated let-7a-3 (methylated) and hypomethylated let-7a-3 (partially methylated or unmethylated). Patients with hypomethylated let-7a-3 had similar responses to standard induction therapy to achieve CR (10/12; 83.3%) compared to those with hypermethylated let-7a-3 (35/48; 72.9%; p = 0.781). Further investigation was performed by using the Kaplan–Meier survival curve and log–rank test to evaluate the suitability of let-7a-3 methylation status as prognostic factors. While patients with favorable karyotypes harbored hypermethylated let-7a-3 less frequently (2/8) than those with intermediate or unfavorable karyotypes (49/61 and 17/18, respectively; Table 2), and further risk-group stratification for favorable karyotypes is less demanding, we focus in assessing the role of let-7a-3 hypermethylation in patients with intermediate and unfavorable karyotypes. With a medium follow-up period of 48 months, let-7a-3 methylation status was not associated with the 5-year survival advantage in all patients

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Table 2 Correlation between let-7a-3 methylation and clinical characteristics of AML patients. Variable Agea <45 ≥45 Gendera Male Female FAB subtypea M0 M1 M2 M3 M4 M5 M6 Karyotypea Favorableb Intermediateb Unfavorableb CEBPA methylationa Hypermethylated Hypomethylated a b

Total (n = 87)

Methylated (n = 68; 78.2%)

Partially methylated (n = 13; 14.9%)

Unmethylated (n = 6; 6.9%)

44 43

32 (72.7) 36 (83.7)

8 (18.2) 5 (11.6)

4 (9.1) 2 (4.7)

45 42

37 (82.2) 31 (73.8)

7 (15.6) 6 (14.3)

1 (2.2) 5 (11.9)

2 18 33 2 23 6 3

2 (100) 17 (94.4) 23 (69.7) 0 (0.0) 19 (82.6) 4 (66.7) 3 (100)

0 (0.0) 1 (5.6) 6 (18.2) 1 (50.0) 3 (13.0) 2 (33.3) 0 (0.0)

0 (0.0) 0 (0.0) 4 (12.1) 1 (50.0) 1 (4.3) 0 (0.0) 0 (0.0)

8 61 18

2 (25.0) 49 (80.3) 17 (94.4)

4 (50.0) 9 (14.8) 0 (0.0)

2 (25.0) 3 (4.9) 1 (5.6)

27 60

17 (63.0) 51 (85.0)

6 (22.2) 7 (11.7)

4 (14.8) 2 (3.3)

P 0.453

0.225

0.181

0.002

0.042

Number of patients (%). Karyotyping subgroups. Favorable: t(15;17), t(8;21), and inv(16); intermediate: normal karyotype and others; unfavorable: −5, −7, +8, and complex.

Fig. 3. Kaplan–Meier survival analysis according to the let-7a-3 methylation status in 60 AML patients receiving standard induction therapy. AML patients were grouped into hypermethylated let-7a-3 and hypomethylated let-7a-3 groups. Five-year survival was analyzed in all patients (a), in patients without a favorable karyotype (b), those with hypomethylated CEBPA (c), and those with hypomethylated CEBPA without a favorable karyotype (d). Number of patients and median survival time are indicated in each subgroup.

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studied (n = 60, p = 0.982; Fig. 3a) or in all patients without the favorable karyotype (n = 50, p = 0.853; Fig. 3b). However, hypermethylated let-7a-3 was associated with longer 5-year survival rates in patients with hypomethylated CEBPA (n = 41; median = 11.3 versus 5.0 months, p = 0.005; Fig. 3c) or even in those with hypomethylated CEBPA without the favorable karyotype (n = 36; median = 11.0 versus 3.6 months, p = 0.016; Fig. 3d).

4. Discussion In this study, we found that let-7a-3 was heavily methylated in AML cell lines and BMMNCs in most AML patients. We also found interesting correlations between let-7a-3 methylation and karyotyping. AML patients with favorable karyotypes harbored methylated let-7a-3 less frequently, while hypermethylated let7a-3 was associated with hypomethylated CEBPA. Additionally, in patients with hypomethylated CEBPA or with hypomethylated CEBPA without a favorable karyotype, hypermethylated let-7a-3 was associated with favorable 5-year survival predictions. In the present study, we assessed the let-7a-3 methylation level using COBRA and bisulphite sequencing. We also proposed a simple method for quantifying let-7a-3 methylation, based on the fact that the methylation percentage of fragment 5 and 6 in let-7a-3 was found to be highly correlated with the overall percentage of let-7a-3 methylation. This approach can be applied to clinical examination to rapidly assess let-7a-3 methylation in which a shorter PCR product only spanning segment 5 and segment 6 can be generated and assessed using a MassARRAY platform or a pyrosequencer. We have previously shown that expression of the tumor suppressor CEBPA is epigenetically controlled, and CEBPA hypermethylation is a favorable prognostic biomarker for AML without CEBPA or NPM1 mutation [8]. The transcription factor C/EBP␣ is essential for early myeloid differentiation. Mutation and epigenetic silencing of CEBPA are both associated with AML [8,24,30,36]. In this study, we demonstrated that let-7a-3 hypermethylation was linked to CEBPA promoter hypomethylation. However, let-7a3 hypermethylation was observed in all 12 patients with CEBPA mutations. The consequence of CEBPA promoter hypomethylation is increased expression of the normal C/EBP␣ levels, which is opposite to the presence of CEBPA mutations. A possible explanation for the conflicting results is that dysregulation of epigenetic control may result from aberrant functioning of DNA methyltransferases, which will affect the expression of more genes comparing to a single gene mutation. Therefore, the results of the correlation analyses may reflect a difference of the cellular context between AML cells with a mutated CEBPA and with an epigenetically silenced CEBPA. We have demonstrated that the methylation of let-7a-3 was inversely correlated with miRNA expression of let-7a. Although this result was obtained from a limited sample size, it is consistent with the evidences from other studies also showing a negative correlation between let-7a-3 methylation and the miRNA expression in normal cells, lung and colon cancer cells, as well as more than 20 samples of ovarian and breast cancer cells [26,27,35]. However, the mechanisms controlling let-7a-3 expression are not fully understood. It is well recognized that NF-␬B pathway activation triggers anti-apoptotic signals to promote tumorigenesis [37]. Recent report revealed that a number of putative NF-␬B-binding sites are identified in the let-7a-3 promoter region, and NF-␬B recruitment primarily is shown to regulate let-7a-3 miRNA expression [38,39]. Since promoter methylation causes silencing of its cognate genes by inhibiting transcription factor binding, further studies are required to examine whether methylation of let-7a3 promoter affects the extent of NF-␬B binding, thus regulating let-7a-3 expression.

The lower incidence of let-7a-3 hypermethylation found in patients with FLT3-TKD mutations indicates a possible correlation between let-7a-3 methylation and FLT3-TKD mutations. However, it is still inconclusive due to the small sample size. Patients with FLT3-TKD mutations present similar clinical features and outcome as those with the more common FLT3-ITD mutations do regarding their higher WBC counts, more BM blasts, and lower OS and DFS comparing to FLT3-ITD/FLT3-TKD double negative patients [40]. However, we did not observe a significant correlation between let-7a-3 methylation and FLT3-ITD mutations. Patients with FLT3-TKD mutations seem to differ in drug response to tyrosine kinase inhibitors [41]. The difference in the mechanism of ligand-independent activation of the FLT3 kinase between these two mutation types should account for the possible correlation between let-7a-3 methylation and FLT3-TKD mutation, and further study with a larger sample size is required to confirm the correlation. It was recently reported that overexpressed let-7a-3 is observed in some AML patients, especially in the female gender. Overexpressed let-7a-3 is linked to poor prognosis in patients who obtained CR. No correlation was found between overexpressed let7a-3 and CR rate or CEBPA mutations, and no analysis was done with FLT3-TKD mutated patients [42]. Our results revealed that there is no correlation between let-7a-3 methylation and gender, while hypermethylated let-7a-3 is correlated with better prognosis in patients with hypomethylated CEBPA. These evidences point to the same direction and suggest that increasing the expression of let7a-3 may excert a negative impact on the survival of AML patients or it may play a leukemogenic role in AML cells in certain subgroup of patients. Let-7 family members target several oncogenes and cell-cycle regulators, imparting tumor-suppressive effects [15–19]. Accordingly, let-7a miRNA is recognized as a tumor suppressor. However, contradictory reports have shown that let-7a overexpression causes oncogenic transformations in a human lung cancer cell line [26], and let-7a-3 methylation is associated with better survival in ovarian cancer patients [27,28], suggesting an oncogenic role. These conflicting findings in different malignancies are likely due to multiple targets silenced by let-7a and also difference in tissue types. In conclusion, we demonstrated the association of let-7a-3 methylation with CEBPA methylation and karyotyping in AML patients. Our results unequivocally support that hypermethylation of let-7a-3 is a positive prognosticator for AML patients with hypomethylated CEBPA. Conflict of interest statement The authors declare no conflict of interest. Author contributions Y-C K and T-C L performed the experiments. H-A H, C-Y C and H-F T contributed patient samples and clinical data. T-C L, W-H F and H-A H performed statistical analysis. Y-C K, W-H F and L-I L analyzed and interpreted the data. Y-C K and L-I Lin wrote the paper. L-I L designed and coordinated the research study. Acknowledgments We thank Mr. Yong-Sian Chen for technique assistance. We thank the faculty and staff in the Division of Hematology at NTUH for referring their AML patients to participate in this study. This work was supported by a research grants from the National Science Council (NSC 96-2320-B-002-039-MY3 and NSC 99-2320-B-002018-MY3), Taiwan.

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