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Leukemia Research 37 (2013) 251– 258

Contents lists available at SciVerse ScienceDirect

Leukemia Research

jo ur nal homep age: www.elsev ier .com/ locate / leukres

xpression analysis of mir-17-5p, mir-20a and let-7a microRNAs and their targetroteins in CD34+ bone marrow cells of patients with myelodysplastic syndromes

iamantina Vasilatoua, Sotirios G. Papageorgioua, Frieda Kontsioti a, Christos K. Kontosb,c,anayiota Tsiotraa, Vassiliki Mpakoua, Maria-Angeliki S. Pavlouc, Christina Economopouloua,eorge Dimitriadisa, John Dervenoulasa, Vasiliki Pappaa,∗

Second Department of Internal Medicine and Research Institute, Athens University Medical School, “Attikon” University General Hospital, Athens, GreeceDepartment of Biochemistry and Molecular Biology, University of Athens, Athens, GreeceLaboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece

r t i c l e i n f o

rticle history:eceived 26 June 2012eceived in revised form6 November 2012ccepted 18 November 2012vailable online 14 December 2012

a b s t r a c t

Mir-17-5p and mir-20a, members of the mir-17-92 family, down-regulate E2F1, which is over-expressedin myelodysplastic syndromes (MDS). Moreover, let-7a down-regulates KRAS, which is aberrantlyexpressed in MDS. We evaluated the expression of the aforementioned microRNAs in CD34+ cells of43 MDS patients using real-time PCR and their target proteins (E2F1, MYC, BCL2, CCND1, and KRAS) byWestern blot. Mir-17-5p and mir-20a were under expressed in high risk MDS patients, compared to lowrisk MDS patients. Similarly, let-7a was under expressed in patients with intermediate or high-risk kary-

eywords:DSicroRNAs

et-7a

otype. Interestingly, there was an inverse correlation between microRNA and the expression levels oftheir targets. Importantly, mir-17-5p and mir-20a constitute favorable prognostic factors in MDS, sincetheir expression was associated with increased overall survival of MDS patients.

© 2012 Elsevier Ltd. All rights reserved.

ir-17-5pir-20a

. Introduction

Myelodysplastic syndromes (MDS) are a group of heteroge-eous disorders of hematopoietic stem cell leading to ineffectiveematopoiesis and variable possibility of leukemic transformation1]. Although several molecular abnormalities have been detectedn MDS [2], the precise steps involved in the initiation and progres-ion of MDS remain unclear.

MicroRNAs are small (19–24 nucleotides), non-coding RNAshat regulate gene expression at the post-transcriptional stage.ince their discovery, microRNAs have been associated with nor-al hematopoiesis and hematopoietic malignancies includingDS [3].The transcription factor E2F1 seems to hold a key role in

isease pathogenesis and progression. E2F1 protein is over-

xpressed in the majority (67%) of patients suffering from MDSut the mechanism of its deregulation is unknown. However,here are data suggesting that E2F1 expression is modified at the

∗ Corresponding author at: Second Department of Internal Medicine and Researchnstitute, Athens University Medical School, “Attikon” University General Hospital,

Rimini Street, Haidari, 12462 Athens, Greece. Tel.: +30 10 583 2549;ax: +30 210 532 6454.

E-mail address: vas pappa@yahoo.com (V. Pappa).

145-2126/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.leukres.2012.11.011

post-transcriptional level [4]. E2F1 is down-regulated by mir-17-5p and mir-20a, members of mir-17-92 family [5]. These data raisequestions about the role of mir-17-5p and mir-20a in a molecu-lar pathway leading to E2F1 overexpression in MDS. Similarly, theanti-apoptotic protein BCL2 is targeted by the mir-17-92 clustersuggesting a possible role of these microRNAs in BCL2 regulationin MDS.

Cyclin D1 (CCND1) induces the expression of miR-17-5p andmiR-20a while miR-17-5p/20a limits the proliferative functionsof CCND1 in breast cancer [6]. In addition, CCND1 and E2F1 areinvolved in intramedullary apoptosis in MDS [7]. These data pro-pose a possible role of CCND1 and E2F1 in MDS pathogenesis.

Another protein family implicated in MDS pathogenesis andprognosis includes the RAS proteins. Activating mutations in RASproteins as well as RAS proteins’ over-expression are associatedwith worse prognosis, and increased probability of leukemic trans-formation [8–11]. RAS proteins are targets of let-7a. This microRNAacts as a tumor suppressor gene and directly represses RAS proteins’expression by binding to their 3′-UTRs [12]. Additionally, it reducestumor growth by inhibiting KRAS gene translation as well as MYC[13,14], a finding that supports the tumor-suppressing role of let-

7a. These data allow us to speculate that RAS deregulation in MDSmay be associated with let-7a expression.

Finally, the BCL2 protein is important in MDS since it suppressesapoptosis and is associated with progression to AML [15].

252 D. Vasilatou et al. / Leukemia Res

Table 1Clinical and biological characteristics of MDS patients.

Number of patients 43Sex (male/female) 34/9

Median (range)

Age (years) 74 (45–87)Hemoglobin (g/dL) 9.6 (7.1–14.0)ANC (×103/mL) 2000 (150–28,000)Platelets (×103/mL) 94,000 (13,000–716,000)BM blasts (%) 6 (2–60)Overall survival (months) 15 (1–45)

N (%)

Cytopenias (43/43 patients)0–1 21 (48.8)2–3 22 (51.2)

Cytogenetic risk group (42/43 patients)Low risk 29 (69.0)Intermediate risk 7 (16.7)High risk 6 (14.3)

WHO type (43/43 patients)RA 12 (27.9)RCMD 6 (13.9)RAEB I 8 (18.6)RAEB II 7 (16.3)AML 8 (18.6)CMML 2 (4.7)

IPSS (40/43 patients)Low risk 12 (30.0)Intermediate risk I 15 (37.5)Intermediate risk II 6 (15.0)High risk 7 (17.5)

WPSS (34/43 patients)Very low risk 8 (23.6)Low risk 4 (11.7)Intermediate risk 9 (26.5)High risk 9 (26.5)Very high risk 4 (11.7)

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A: refractory anemia; RCMD: refractory cytopenia with multilineage dysplasia;AEB: refractory anemia with excess blasts; AML: acute myeloid leukemia; CMML:hronic myelomonocytic leukemia.

Taking into consideration the role of E2F1, KRAS, BCL2, CCNDnd MYC in MDS pathogenesis and leukemic transformation, andheir regulation by mir-17-5p, mir-20a and let-7a, these microR-As were selected for expression analysis. More specifically, theurpose of the current study is to analyse the expression status ofir-17-5p, mir-20a and let-7a, and their targets (E2F1, KRAS, BCL2,

CND1, MYC) in CD34+ cells of MDS patients and to associate theseith prognostic factors and survival.

. Materials and methods

.1. Patients and control group

We collected CD34+ cells from BM samples of 43 patients with MDS and CD34+ells of peripheral blood from 18 healthy donors for hematopoietic stem cell trans-lantation (HSCT), who served as a control group (median age: 55 years). Theollection of CD34+ cells from BM was taken at diagnosis prior to the initiationf any therapy.

The current study was performed in accordance with the ethical standardsf the World Medical Association Declaration of Helsinki (version: 2008), andas approved by the institutional review board of University General Hospital

Attikon” (Athens, Greece). Moreover, written informed consent was obtained fromll patients and healthy volunteers participating in the current study.

Diagnosis of MDS was established according to the WHO recommended criteria.he risk stratification of patients was determined according to the Internationalrognostic Score System (IPSS) and WHO Prognostic Score System (WPSS). Patients’linical characteristics as well as their classification according to IPSS and WPSS areemonstrated in Table 1.

.2. Sample collection and CD34+ cell isolation

Samples from BM of MDS patients and peripheral blood (PB) of CD34+ells healthy donors for HSCT were collected in tubes containing 2 mM EDTA.

earch 37 (2013) 251– 258

Mononuclear cells (MNCs) were isolated using Fiqoll-Paque and CD34+ cells werepositively selected using CD34 Microbead kit (Miltenyi Biotec, Cambridge, MA,USA). Prior to preparing RNA from CD34+ cells, their purity was verified using flowcytometry. CD34+ cells comprise more than 90% of the isolated cells.

2.3. RNA isolation and cDNA synthesis

Isolation of small RNA-containing total RNA from CD34+ cells and its reversetranscription into cDNA were performed as described in supplementary data.

2.4. Quantitative real-time PCR (qRT-PCR)

Quantitative real-time PCR was performed using a TaqMan microRNA assay,as described in supplementary data. Each real-time PCR reaction was performed induplicate, in order to evaluate data reproducibility. Threshold cycle values were thencalculated by determining the point at which the emitted fluorescence exceeded thethreshold [16]. Calculations were made using the comparative CT (2−��CT) method.The prerequisites for the application of the 2−��CT method were checked in a vali-dation experiment, in which CT values of mir20a, mir17-5p, let7a, and RNU48 weremeasured in a dilution series K-562 cDNA over a 104-fold range and then plottedagainst log cDNA dilution. Normalized results were expressed as the ratio of eachtarget (mir20a, mir17-5p, and let7a) miRNA copies to 1000 RNU48 snoRNA copies(c/Kc), calculated for each specimen, in relation to the same ratio calculated forK-562 cells.

2.5. Protein extraction and Western blot analysis

Total protein was extracted from CD34+ cells, and Western blot analysisfor ACTIN, MYC, E2F1, CCND1, BCL2, and KRAS was performed as described insupplementary data.

2.6. Statistical analysis

Since the distributions of mir20a, mir17-5p and let7a expression levels in MDSpatients were not Gaussian, the analysis of the differences between the groups wasperformed with the non-parametric Mann–Whitney U test or Kruskal–Wallis test,where appropriate. For categorization of mir20a, mir17-5p and let7a expression lev-els, the X-tile algorithm was used to generate an optimal cutpoint [17]. This processproduced an optimal cutoff of 156.35 c/Kc for mir-20a expression levels, which isequal to the 73th percentile, and 63.20 c/Kc for mir-17-5p expression levels, whichis equal to the 73th percentile. Following the same procedure for let-7a expression,the optimal cutoff of 15234.90 c/Kc, equal to the 80th percentile, was generated.

According to the aforementioned cutoffs, mir20a, mir17-5p and let7a miRNAexpression values were classified as positive or negative. Associations of mir-20a,mir-17, and let-7a status with categorical clinicopathological variables of MDSpatients were analyzed using either the chi-square (�2) or the Fisher’s exact test,where appropriate. Relationships between different continuous variables were alsoassessed by Spearman correlation coefficient (rs).

In order to assess the association between the prognostic markers and the rel-ative risk for death of patients, we developed Cox proportional hazard regressionmodels. Cox univariate regression analysis discloses the strength of the correlationbetween each clinicopathological parameter and overall survival (OS). Follow-upinformation was available for 41 patients. Moreover, multivariate regression mod-els were developed for the expression of each studied miRNA and were adjusted forIPSS and WPSS. Only 33 patients, for whom the status of all variables was known,were included in the Cox multivariate analysis.

Survival analysis was also performed by constructing Kaplan–Meier OS curves,where differences between curves were evaluated by the log-rank (Mantel–Cox)test. OS was calculated from the date of diagnosis to the date of death of any causeor last follow-up. The level of significance was defined at a probability value of lessthan 0.05 (p < 0.05).

3. Results

3.1. Mir-17-5p, mir-20a, and let-7a expression analysis in MDSpatients and healthy controls

Expression analysis of mir-17-5p, mir-20a, and let-7a in CD34+cells isolated from BM of 43 MDS patients and from PB of 18healthy donors did not reveal any significant differences betweenthese two groups (Table 2). However, mir-17-5p expression wassignificantly different among patients belonging to the low orintermediate I IPSS risk group, patients of the intermediate II or

high risk group, and healthy controls (p = 0.036; Fig. 1A). Interest-ingly, mir-17-5p levels were also significantly lower in patientswith intermediate or high cytogenetic risk than in healthy controls(p = 0.016; Supplementary Table 1). On the other hand, differences

D. Vasilatou et al. / Leukemia Research 37 (2013) 251– 258 253

Table 2MiR-17-5p, miR-20a, and let-7a microRNA expression analysis in MDS patients and healthy controls.

Variable Mean ± SE Range Percentiles

25th 50th 75th

Median

Mir-17-5p expression (c/Kc) in MDS patients (N = 43) 43.82 ± 7.19 0.005–143.90 4.25 24.75 65.15Mir-17-5p expression (c/Kc) in healthy controls (N = 18) 44.41 ± 11.35 1.47–133.61 7.76 27.68 77.39

p = 0.800a

MiR-20a expression (c/Kc) in MDS patients (N = 43) 106.15 ± 17.78 0.003–410.38 1.11 62.94 174.34MiR-20a expression (c/Kc) in healthy controls (N = 18) 70.70 ± 18.53 0.12–211.69 7.64 39.92 156.22

p = 0.647a

Let-7a expression (c/Kc) in MDS patients (N = 43) 7304.2 ± 1127.6 53.29–25219.0 1536.9 4228.1 11157.9Let-7a expression (c/Kc) in healthy controls (N = 18) 7413.9 ± 1666.8 139.7–19716.6 2337.3 3680.4 13857.1

p = 0.831a

c

iIr(vt

FirKpte

/Kc: miRNA copies/1000 RNU48 snoRNA copies; SE: standard error of the mean.a Calculated using the Mann–Whitney U test.

n the expression profile of mir-20a among low or intermediate IPSS risk MDS patients, patients of the intermediate II or high

isk group, and normal controls were marginally insignificantp = 0.053; Fig. 1B), whereas mir-20a expression in patients withery low or low WPSS risk was significantly higher, comparedo the normal control group (p = 0.031; Supplementary Table 1).

ig. 1. Comparison of the distribution of mir-17-5p (A) and mir-20a (B) expressionn low or intermediate I IPSS risk MDS patients, patients of the intermediate II or highisk group, and normal controls. P values were calculated using the non-parametricruskal–Wallis test. The bottom and top of each box represent the 25th and 75thercentile, respectively, while the bold band near the middle of each box indicateshe 50th percentile (the median value) of each group. Whiskers show the mostxtreme values.

Finally, no statistically significant differences were found amongall WPSS-based subgroups of MDS patients and healthy donors(Supplementary Fig. 1). A complete list of the values of miRNAexpression in healthy controls and MDS patients, grouped accord-ing to BM blast percentage, cytogenetic risk, WHO classification,IPSS and WPSS risk are presented in Supplementary Table 2.

3.2. Comparison of mir-17-5p, miR-20a, and let-7a expressionstatus among subgroups of MDS patients

The expression of miR-17-5p and miR-20a was remarkablydifferent among distinct subgroups of MDS patients (Table 3).Interestingly, patients with less than 10% blasts in the BM had sig-nificantly higher levels of miR-17-5p, compared to patients withmore than 10% blasts (p = 0.023). Furthermore, patients with lowrisk karyotype presented a much stronger expression of the abovementioned microRNAs compared to patients with intermediate orhigh risk karyotype (p = 0.018 and p = 0.001 respectively). Regardinglet-7a, patients with low risk karyotype showed a much strongerexpression of this microRNA compared to patients with interme-diate or high risk karyotype (p = 0.035). A statistically significantincrease in miR-17-5p levels was also observed in patients suffer-ing from RA or RCMD, compared to RAEB-I or RAEB-II and to AMLpatients (p = 0.049). Additionally, mir-17-5p and mir-20a were ele-vated in patients belonging to the low or intermediate I IPSS riskgroup, compared to patients of the intermediate II or high risk group(p = 0.011 and p = 0.025, respectively).

As presented in Table 4, of the 43 MDS cases examined, 11(25.6%) were classified as positive for mir-17-5p and miR-20aexpression and 32 (74.4%) as negative, while 8 (20%) were let-7apositive and 35 (80%) were let-7a negative (Section 2.6). Significantnegative associations were observed between mir-17-5p expres-sion and the percentage of blasts in the patients’ BM (p = 0.008),cytogenetic risk (p = 0.009) and IPSS risk group (p = 0.007). Mir-20aexpression was also found to be negatively associated with IPSSrisk category, as patients belonging to the low or intermediate Irisk group were more frequently mir-20a-positive, compared topatients with intermediate II or high risk disease (p = 0.016). Finally,a negative association was found between let-7a expression andthe percentage of BM blasts (p = 0.036), risk of disease as deter-mined by cytogenetics (p = 0.043), WHO classification (p = 0.020),IPSS (p = 0.037), and WPSS (p = 0.033).

3.3. Correlations between microRNA levels and expression of

targeted proteins involved in MDS pathogenesis

In MDS patients, a strong positive correlation was foundbetween mir-17-5p and mir-20a expression (rs = 0.807, p < 0.001).

254 D. Vasilatou et al. / Leukemia Research 37 (2013) 251– 258

Table 3MiR-17-5p, miR-20a, and let-7a microRNA expression analysis in subgroups of MDS patients.

Variable Mean ± SE Median Range P value

MiR-17-5p expression (c/Kc) in patients withBM blasts (%) ≤10 (N = 28) 57.28 ± 9.83 43.67 0.005–143.90 0.023a

BM blasts (%) >10 (N = 15) 18.68 ± 5.23 13.79 0.058–62.94

MiR-17-5p expression (c/Kc) in patients withLow cytogenetic risk (N = 29) 59.34 ± 9.26 48.03 0.030–143.90 0.001a

Intermediate or high cytogenetic risk (N = 13) 11.49 ± 3.81 8.37 0.005–39.28

MiR-20a expression (c/Kc) in patients withLow cytogenetic risk (N = 29) 128.69 ± 22.02 86.57 0.003–410.38 0.018a

Intermediate or high cytogenetic risk (N = 13) 63.87 ± 28.69 0.324 0.016–348.69

Let-7a expression (c/Kc) in patients withLow cytogenetic risk (N = 29) 8985.9 ± 1488.0 5897.1 92.1–25219.0 0.035a

Intermediate or high cytogenetic risk (N = 13) 3996.3 ± 1243.2 2234.6 53.3–14621.3

MiR-17-5p expression (c/Kc) in patients withRA or RCMD (N = 18) 58.63 ± 13.10 38.05 0.030–143.90 0.049b

RAEB I or RAEB II (N = 15) 44.13 ± 10.87 34.44 0.077–125.61AML (N = 8) 11.80 ± 4.69 10.62 0.005–39.28

MiR-17-5p expression (c/Kc) in patients withLow or intermediate I IPSS risk (N = 27) 59.40 ± 9.96 45.44 0.030–143.90 0.011a

Intermediate II or high IPSS risk (N = 13) 18.75 ± 6.16 8.37 0.005–62.94

MiR-20a expression (c/Kc) in patients withLow or intermediate I IPSS risk (N = 27) 129.71 ± 23.83 103.67 0.003–410.38 0.025a

Intermediate II or high IPSS risk (N = 13) 43.37 ± 14.92 29.77 0.018–153.89

c/Kc: miRNA copies/1000 RNU48 snoRNA copies; SE: standard error of the mean.

Lvpbpp

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p

a Calculated using the Mann–Whitney U test.b Calculated using the Kruskal–Wallis test.

evels of both mir-17-5p and mir-20a were also shown to co-ary with let-7a, yet moderately (rs = 0.491, p = 0.001 and rs = 0.459,

= 0.002, respectively). No significant correlations were foundetween the studied microRNAs and continuous variables of MDSatients such as hemoglobin, absolute neutrophil count, number oflatelets, and percentage of BM blasts.

In our study, the expression analysis of proteins that are impli-ated in the pathobiology of MDS revealed that the transcription

actor E2F1 and the anti-apoptotic protein BCL2 correlate stronglyn a negative manner with mir-17-5p and mir-20a, while a statis-ically significant, strong negative correlation was also observedetween KRAS protein expression and let-7a levels (Table 5).

able 4ssociations between miR-17-5p, miR-20a, let-7a status, and clinicopathological variable

Variable Total No. patients (%) P value N

Mir-17-5pnegativea

Mir-17-5ppositivea

Mn

BM blasts≤10% 28 17 (60.7) 11 (39.3) 0.008b 1>10% 15 15 (100.0) 0 (0.0) 1

Cytogenetic risk groupLow risk 29 18 (62.1) 11 (37.9) 0.009b 1Intermediate or high risk 13 13 (100.0) 0 (0.0) 1

WHO classificationRA or RCMD 18 11 (61.1) 7 (38.9) 0.091c 1RAEB I or RAEB II 15 12 (80.0) 3 (20.0) 1AML 8 8 (100.0) 0 (0.0)

IPSSLow or intermediate I risk 27 16 (59.3) 11 (40.7) 0.007b 1Intermediate II or high risk 13 13 (100.0) 0 (0.0) 1

WPSSVery low or low risk 12 6 (50.0) 6 (50.0) 0.065c

Intermediate risk 9 6 (66.7) 3 (33.3)

High or very high risk 13 12 (92.3) 1 (7.7) 1

a Cutoff point: 63.20 c/Kc for miR-17-5p, equal to the 73rd percentile; 156.35 c/Kc foercentile.b Calculated by Fisher’s exact test.c Calculated by chi-square (�2) test.

Furthermore, CCND1 and mir-17-5p were adversely related toeach other, yet this correlation was not proven to be statisticallysignificant (rs = −0.588, p = 0.074). MicroRNA and protein levels ofrepresentative samples are shown in Supplementary Fig. 1.

3.4. Association of mir-17-5p, mir-20a, and let-7a expressionstatus with OS of MDS patients

Out of 41 patients for whom follow-up information was avail-able, 18 patients (43.9%) died during the respective follow-upperiods, due to causes related to MDS. The median OS was 15months (range: 1–45 months). In Cox univariate survival analysis

s.

o. patients (%) P value No. patients (%) P value

ir-20aegativea

Mir-20apositivea

Let-7anegativea

Let-7apositivea

8 (64.3) 10 (35.7) 0.0654 20(71.4) 8(28.6) 0.036b

4 (93.3) 1 (6.7) 15(100.0) 0(0.0)

9 (65.5) 10 (34.5) 0.134 21(72.4) 8(27.6) 0.043b

2 (92.3) 1 (7.7) 13(100.0) 0(0.0)

0 (55.6) 8 (44.4) 0.0795 11(61.1) 7(38.9) 0.020c

3 (86.7) 2 (13.3) 14(93.3) 1(6.7)7 (87.5) 1 (12.5) 8(100.0) 0(0.0)

7 (63.0) 10 (37.0) 0.0164 19(70.4) 8(29.6) 0.037b

3 (100.0) 0 (0.0) 13(100.0) 0(0.0)

6 (50.0) 6 (50.0) 0.0655 8(66.7) 4(33.3) 0.033c

6 (66.7) 3 (33.3) 5(55.6) 4(44.4)2 (92.3) 1 (7.7) 13(100.0) 0(0.0)

r miR-20a, equal to the 73rd percentile; 15234.9 c/Kc or let-7a, equal to the 80th

D. Vasilatou et al. / Leukemia Research 37 (2013) 251– 258 255

Table 5Relationships between the miRNA levels and their target-protein levels in MDSpatients.

MYC KRAS E2F1 CCND1 BCL2

Mir-20ars −0.140a 0.292a −0.632a −0.188a −0.888a

P 0.700b 0.413b 0.050b 0.602b 0.001b

MiR-17-5prs −0.006a 0.503a −0.758a −0.588a −0.806a

P 0.987b 0.138b 0.011b 0.074b 0.005b

Let-7ars 0.139a −0.794a 0.418a 0.309a 0.055a

P 0.701b 0.006b 0.229b 0.385b 0.881b

a Spearman’s rank correlation coefficient.b Calculated using Spearman correlation.

Table 6miRNA expression and overall survival (OS) of MDS patients.

Variable Overall survival

HR 95% CI P value

Mir-20aNegative 1.00Positive 0.128 0.017–0.966 0.046

Mir-17-5pNegative 1.00Positive 0.118 0.016–0.888 0.038

Let-7aNegative 1.00Positive 0.187 0.025–1.41 0.104IPSS (ordinal) 8.18 2.77–24.16 <0.001WPSS (ordinal) 4.17 1.65–10.59 0.003

Hc

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ltWsi

4

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Fig. 2. Kaplan–Meier curves for overall survival (OS) of MDS patients. mir-17-5pand mir-20a expression status possess a favorable prognostic value in MDS, as mir-17-5p-positive patients (A) and/or mir-20a-positive patients (B) have significantly

R: hazard ratio, estimated from Cox proportional hazard regression model; CI:onfidence interval of the estimated HR.

Table 6), the risk of death was shown to be significantly relatedo mir-17-5p and mir-20a expression. Therefore, in addition toPSS and WPSS that were confirmed as significant predictors ofFS (p < 0.001 and p = 0.003, respectively), mir-17-5p and mir-20axpression were shown to predict better OS in MDS, since mir-7-5p-positive and mir-20a-positive patients were at lower risk ofeath (HR = 0.118, 95% CI = 0.016–0.888, p = 0.038 and HR = 0.128,5% CI = 0.017–0.966, p = 0.046, respectively). On the other hand,

et-7a expression did not prove to be a statistically significant prog-ostic factor in MDS (p = 0.104).

In order to evaluate mir-17-5p, mir-20a, and let-7a expres-ion in terms of predicting survival outcome, we also performedaplan–Meier survival analysis. In accordance with the afore-entioned results, Kaplan–Meier analysis revealed significantly

ncreased OS of mir-17-5p-positive MDS patients, as comparedo mir-17-5p-negative patients (p = 0.011) (Fig. 2A). Similar to

ir-17-5p, mir-20a expression is a favorable marker in MDS,s mir-20a-positive patients had a higher survival probabilityhan mir-20a-negative patients did (p = 0.016) (Fig. 2B). Regard-ng let-7a, no statistically significant difference was found betweenaplan–Meier OS curves of let-7a-positive and -negative MDSatients (p = 0.063) (Fig. 2C).

In the multivariate survival analysis, mir-17-5p, mir-20a, andet-7a expression was not found to add any prognostic power inhe developed Cox regression model when adjusted for IPSS and

PSS (data not shown). Hence, these three miRNAs were nothown to constitute independent predictors of favorable prognosisn MDS.

. Discussion

We evaluated the expression levels of three microRNAs, mir-7-5p, mir-20a and let-7a and their target proteins in CD34+

longer OS (p = 0.011 and p = 0.016, respectively), compared to mir-17-5p-negativeand/or mir-20a-negative MDS patients, respectively. Regarding let-7a expressionstatus (C), it prognostic potential was marginally insignificant (p = 0.063).

cells from BM of patients suffering from MDS. Firstly, the afore-mentioned microRNAs showed a particularly different expression

profile between high and low risk MDS, secondly, microRNAexpression was related to the expression levels of their targets

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56 D. Vasilatou et al. / Leukem

nd finally, we found a correlation between patient survival andicroRNA expression levels.Previous studies have tried to evaluate the role of microRNA

xpression in the pathogenesis and prognosis of MDS, but the exactathways involved in this group of hematological malignanciesemain unclear [18]. Erdogan et al. focused on low risk MDS anderformed microarray and RT-PCR analysis of microRNA expres-ion in mononuclear cells (MNC). They identified 13 microRNAsifferentially expressed between patients and controls [19]. Hus-ein et al. used low-density RT-PCR based array and found differenticroRNA signature between patients with normal karyotype and

hose with chromosomal alteration [20]. Pons et al. used RT-PCRo compare the expression of microRNAs in MNC from PB and BMetween patients and controls and found 12 microRNAs differen-ially expressed [21]. Finally, Sokol et al. focused on the prognosticalue of microRNA expression in MDS [22] and Merkerova et al.sed illumina array platform and demonstrated that 22 microRNAsistinguished between patients and controls and MDS subtypes23]. However, the exact role of mir-17-5p, mir-20a and let-7a inD34+ cells of patients with low and high risk MDS had not beenlarified.

We found that mir-17-5p and mir-20a were down-regulatedn high risk MDS (i.e. MDS with intermediate II and highPSS/intermediate or high risk karyotype) compared to low risk

DS (i.e. low and intermediate I IPSS/low risk karyotype). Ofote, mir-17-5p expression correlated with mir-20a expression,hich is compatible with the fact that they come from the same

enetic locus (13q31). Other groups have previously found dis-inct expression pattern between low and high risk MDS [22,23].owever, the expression of mir-17-5p and mir-20a did not dif-

er at a statistically significant level in these studies. It has beenhown that mir-17-92 family and especially mir-17-5p and mir-0a block monocytic differentiation through suppression of theroto-oncogene AML1 [24–26]. Furthermore, mir-17-92 cluster isver-expressed in CD34+ cells of primary chronic myeloid leukemiaCML), but not in blast crisis [27] and in MLL rearranged AML by

odulating p21 expression [28–30]. Our results reveal a differentxpression status of mir-17-5p and mir-20a in low and high riskDS. Taken together these data suggest a different role of mir-17-

p and mir-20a in different stages of myeloid malignancies.Interestingly, mir-20a showed a bimodal expression pattern,

ince it is up-regulated in patients with low IPSS, compared toontrols, and down-regulated in patients with high IPSS. Mir-17-p expression was significantly lower in patients with high IPSSompared to controls but its expression levels did not differ at a sta-istically significant level between low IPSS MDS and controls. Ouresults agree with previous studies that have also found members ofir-17-92 cluster, mir-17-3p and mir-17-5p, to be over-expressed

n low risk MDS compared to controls [21]. The different expressionf microRNAs between controls, low risk and high risk MDS prob-bly could reflect differences in cell proliferation rates betweenormal BM, low risk MDS and high risk MDS, since it is well knownhat low risk MDS shows high rates of apoptosis whereas in highisk MDS proliferation predominates. Nevertheless, regarding theifferences in the expression pattern of these microRNAs observedetween MDS patients and normal controls, it should be added thathey could be also due to the different source of cells (BM from

DS patients and PB from controls), which might bring inaccura-ies in the comparison of expression between patients and healthyonors.

In our study, mir-17-5p and mir-20a expression was inverselyorrelated with the expression of their target, E2F1. E2F1 is known

or its role in cell proliferation and apoptosis [31,32]. It holds aey role in G1/S transition and apoptosis through p53 dependent33] and independent pathway [34]. Its ectopic expression has beenssociated with apoptosis establishing E2F1 as a tumor suppressor.

earch 37 (2013) 251– 258

However, several studies support its role as an oncogene as well[35]. As controversial as it may seem, E2F1 can act both as an onco-gene and as tumor suppressor gene depending on the genetic ormolecular background of the cell [32]. E2F1 is able to induce tran-scription of both S-phase and pro-apoptotic genes. In the light ofthese results, it is tempting to speculate that de-repression of E2F1expression, due to mir-17-5p and mir-20 down-regulation, bringsabout apoptosis with concomitant S-phase changes, a process com-patible with the signal antonymy theory [36].

Additionally, mir-17-5p and mir-20a expression inversely cor-related with the expression of BCL2, which in turn suggests thatpatients with high risk MDS expressed higher levels of BCL2 thanlower risk MDS. Our result is compatible with previous studiesdemonstrating that this anti-apoptotic protein is implicated in theleukemic evolution of MDS [15].

Expression analysis of let-7a revealed that its under-expressionis associated with high risk MDS (intermediate II or highIPSS/intermediate or high risk karyotype). Previous studies haveassociated let-7a with the pathogenesis and worse prognosis ofsolid tumors [37–40] and hematological malignancies, such as AML[41]. Our results are in accordance with other studies showingthat microRNAs of the let-7 family (let-7e) are down-regulatedin MDS compared to controls [22]. Furthermore, we found thatRAS protein levels negatively correlated with let-7a levels. Thereare several lines of evidence supporting that mutated RAS pro-teins are constantly activated leading to worse prognosis andincreased probability of AML transformation. Over-expression ofRAS proteins probably suggests another possible mechanism of RASderegulation. Taken together these data propose a new molecularpathway of high risk MDS and MDS-related AML, where low levelsof let-7a result in loss of control of RAS proteins’ expression andactivation of the kinase cascade that transfers oncogenic signals tothe cell nucleus.

Finally, it has been proven that EGFR/RAS/RAF pathwaypromotes E2F1-induced cell proliferation by suppressing E2F1-induced apoptotic pathways [42–44]. As a result, we can assumethat mir-17-5p and mir-20 down-regulation in high risk MDS leadsto E2F1 de-repression and that RAS over-expression forces, cell toproliferate, a process known to characterize high risk MDS.

Of note, survival analysis showed that the expression of mir-17-5p and mir-20a was associated with significantly better OS andtherefore represents a favorable prognostic factor. As mentionedabove mir-17-5p and mir-20a expression was negatively associ-ated to established prognostic factors such as IPSS as well as to theexpression of E2F1, a factor associated with poor prognosis in MDS.Mir-17-92 expression has been associated with disease prognosisand survival in other hematological malignancies such as multi-ple myeloma [45]. In conclusion, these data suggest that mir-17-5pand mir-20a could represent prognostic factors in MDS patients.However, further studies will shed light on the exact role of thesemicroRNAs in MDS prognosis.

Although let-7a expression was not significantly associatedwith OS, patients positive for let-7a tend to have better OS com-pared to let-7a negative patients (p = 0.063). However, Zuo et al.have previously shown that circulating let-7a predicts OS andprogression-free survival (PFS) in MDS patients and that patientswith low circulating let-7a levels have better OS than patients withhigh levels of circulating let-7a [46]. This discrepancy could beattributed to the fact that we measured let-7a expression levelsin CD34+ cells while Zuo et al. measured circulating levels of themicroRNA due to reduced export of microRNAs from intracellu-lar to extracellular environment. Differences between intracellular

and extracellular profile of let-7a have been also reported in gastriccancer cell lines [47]. Finally, the different profile of intracellularand extracellular microRNAs could be the result of a cellular selec-tive mechanism or microRNA secretion [48]. There is no doubt that

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ore studies are needed to confirm the prognostic value of let-7an MDS BM and PB.

In conclusion, our study demonstrates that microRNA expres-ion has a key role in MDS prognosis. The identification of distinctxpression patterns of mir-17-5p, mir-20a and let-7a between lownd high risk MDS suggest that these microRNAs could be incorpo-ated in the future in new prognostic models aiming at selectingatients where intensification of treatment is needed. The cor-elation between the expression of microRNAs and their targetsuggests the possible pathway through which microRNAs serveheir role in disease progression and AML transformation. The prog-ostic utility of mir-17-5p and mir-20a was confirmed by the facthat their expression correlated with a better OS representing aavorable prognostic factor. However, these microRNAs do not rep-esent independent prognostic factors. Undoubtedly, WPSS andPSS represent stronger prognostic factors in MDS. Further investi-ations should be carried out in large groups of patients in order tostablish the prognostic role of these microRNAs in clinical practice.

onflict of interest statement

The authors have no conflict of interest to disclose.

cknowledgments

Funding. This work has been supported by a grand from theellenic Co-operative Oncology group (HeCOG) and the researchrogram of Kapodistrias.

Contributions. D.V. designed the study, analyzed data, performedhe experiments and wrote the paper. S.G.P. designed the study,nalyzed data and wrote and reviewed the paper. F.K. designedhe study, participated in experiments and provided technicalupport. C.K.K. participated in experiments, performed the sta-istical analysis and drafted parts of the paper. P.T. designed thetudy and provided technical support. V.M. provided technical sup-ort. MA.S.P. participated in experiments. C.E. collected the clinicalata of the patients. G.D. reviewed the paper. J.D. designed andeviewed the paper. V.P. designed the study, analyzed data, wrotend reviewed the paper.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.leukres.2012.11.011.

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