Diagnostic potential of PTEN-targeting miR-214 in the blood of breast cancer patients

PRECLINICAL STUDY

Diagnostic potential of PTEN-targeting miR-214in the blood of breast cancer patients

Heidi Schwarzenbach ?Karin Milde-Langosch

?

Bettina Steinbach ?Volkmar Mu

¨ller ?Klaus Pantel

Received:25November 2011/Accepted:7February 2012óSpringer Science+Business Media,LLC.2012

Abstract MicroRNAs play a role in breast cancer devel-opment and progression by post-transcriptional repression of the expression of important genes,such as the tumor suppressor gene phosphatase and tensin homolog (PTEN).The focus of the current study was to examine the diag-nostic potential of circulating cell-free microRNAs target-ing PTEN in breast cancer.Our analyses were performed on preoperative serum samples of 102patients with early breast cancer and a subset of 34postoperative samples,as well as of 32patients with benign breast disease and 53healthy women.The relative concentrations of four circu-lating microRNAs (miR-19a,miR-20a,miR-21,and miR-214)in blood serum were measured by TaqMan MicroRNA assays.Levels of preoperative serum miR-20a and miR-21were signi?cantly higher in patients with breast cancer and benign disease than in healthy women (p =0.0001),but only serum miR-214could discriminate malignant from benign tumors and healthy controls (p =0.0001)with an area under the curve of 0.878and 0.883in ROC analysis,respectively.Moreover,miR-214levels signi?cantly decreased in the postoperative serum samples (p =0.0001)as compared to the preoperative samples.The comparison with the clinicopathologic data of the breast cancer patients

showed that increased miR-214levels were associated with a positive lymph node status (p =0.039).Our data show that circulating,cell-free miR-214has diagnostic potential in breast cancer as indicator of malignant disease and metastatic spread to regional lymph nodes.Since PTEN is an important target gene of miR-214,this ?nding could also have potential implications for therapeutic approaches.Keywords Serum miRs áTumor progression áLymph node metastasis áPTEN

Introduction

Development of breast cancer and its metastases are complex multistep processes associated with numerous genetic alterations,downregulation of tumor suppressor genes,upregulation of oncogenes,and early hematoge-neous dissemination of tumor cells [1–3].Besides inacti-vation of BRCA1/2and p53,repression of the tumor suppressor gene phosphatase and tensin homolog (PTEN)plays also a role in the predisposition for breast cancer [4].PTEN encodes a dual protein/lipid phosphatase and its main substrate is phosphatidyl-inositol 3,4,5triphosphate (PIP3),the product of the phosphoinositide 3-kinase (PI3K).PTEN negatively controls the intracellular PI3/AKT signal transduction pathway regulating the signaling of multiple biological processes,such as cell metabolism,proliferation,and apoptosis [5].Repression of PTEN results in the activation of the PI3K-Akt pathway,which gives rise to increased cell survival,may cause malignant transformation and has been associated with the invasive-ness and metastasis of breast cancer [6].

MicroRNAs (miRs)are small,non-coding RNA mole-cules consisting of approximately 22nucleotides.They

Electronic supplementary material The online version of this article (doi:10.1007/s10549-012-1988-6)contains supplementary material,which is available to authorized users.

H.Schwarzenbach (&)áB.Steinbach áK.Pantel

Department of Tumor Biology,University Medical Center Hamburg-Eppendorf,Martinistra?e 52,20246Hamburg,Germany

e-mail:hschwarz@uke.uni-hamburg.de

https://www.360docs.net/doc/bc15000643.html,de-Langosch áV.Mu

¨ller Clinic of Gynecology,University Medical Center Hamburg-Eppendorf,Hamburg,Germany

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DOI 10.1007/s10549-012-1988-6

modulate post-transcriptionally the expression of numerous genes,such as tumor suppressor genes,by binding sequence-speci?cally to their target mRNA and inhibiting their translation into proteins or degrading the mRNA[7]. MiRs are involved in the regulation of different cellular processes,e.g.,apoptosis,hematopoietic cell differentia-tion,metabolism,neural development,and metastasis[8]. The importance of miRs in the regulation of these pro-cesses has been documented by the production of knockout mice.Knocking out the enzyme Dicer,responsible for the maturation of miRs,caused lethality of mouse embryos[9]. As half of human miRs are located in fragile chromosomal regions harboring DNA ampli?cations,deletions or trans-locations,their expression is frequently deregulated during tumor development.In tumors their changed expression leads to oncogenic function[10].

Apart from binding to numerous mRNAs,the miR-19a, miR-20a,miR-21,and miR-214have reported to have a common target being PTEN[11].Knockdown of miR-19a by transfection with anti-miR-19a showed a positive effect in bladder cancer cells by inhibiting cell growth,inducing cell apoptosis and activating the PTEN/Akt pathway[12]. During neuronal lineage differentiation miR-20a modulated the intracellular activity of transcription factor E2F by tar-geting the30-untranslated region(UTR)of PTEN[13].In breast cancer cells the estradiol-mediated decrease in miR-21correlated with increased protein expression of PTEN. Transfection of MCF-7cells with antisense to miR-21 mimicked the estradiol-induced increase in PTEN[14].In patients with ovarian cancer,miR-214signi?cantly induced cell survival and cisplatin resistance through targeting PTEN and consequently the PI3K/AKT pathway[15].

Apoptotic and necrotic cells of the primary tumor dis-charge their nucleic acids,such as DNA and RNA,into the blood circulation.Numerous publications have reported on the elevated levels of circulating nucleic acids in blood of breast cancer patients[16].However,in the year2008, circulating miRs were for the?rst time described and detected in the serum of patients with diffuse B-cell lym-phoma[17].Protected from the degradation by endogenous RNase activity,miRs circulate in a remarkably stable form in human blood[18].Recently,high circulating miR con-centrations were detected in serum of breast cancer patients which signi?cantly correlated with the clinicopathologic parameters of these patients[16].Serum is easy to obtain also at different time points during the course of the dis-ease.Thus,circulating cell-free miRs may have potential as diagnostic and prognostic biomarkers.

In the current study,we assessed the serum levels of cir-culating PTEN-targeting miRs in the blood serum of patients with early breast cancer in comparison to patients with benign breast disease and healthy women,in order to eval-uate the diagnostic potential of these cell-free nucleic acids.Materials and methods

Patient design and healthy women

A total of102female patients with early breast cancer (mean age:60,range:30–82),and32patients with benign breast disease(mean age:50,range:26–63)who received treatment in the Clinic of Gynecology,University Medical Center Hamburg-Eppendorf from July1999to February 2006were recruited.All histological investigations con-?rmed pathologic?ndings of invasive breast cancer or benign?ndings.Metastatic spread in breast cancer patients was excluded by chest radiology,liver ultrasound,and bone scan.The peripheral blood was obtained before any surgery and chemotherapy,radiotherapy or pharmacother-apy.Follow-up samples from34breast cancer patients were obtained3–8months after surgery.Table1summa-rizes the clinical and histopathological parameters of the patients with breast cancer.Patients with benign breast tumor had either a?broadenoma(57%)or mastopathy (43%).In addition,peripheral blood samples from53 healthy women with no history of cancer and in good health based on self-report were enrolled(mean age:50, range:20–79).All patients and healthy controls gave their informed consent.The examination of samples was approved by the local ethics review boards.

Extraction of total RNA

Ten ml whole blood of each patient were collected in routine S-Monovette tubes(Sarstedt,Nu¨mbrecht,Ger-many)and immediately stored at4°C.The preparation of serum was performed within4h,stored at-80°C and described elsewhere[19].For isolation of total RNA from the serum samples,the mirVana PARIS kit(Ambion, Darmstadt,Germany)was used.Four hundred l L of serum samples were incubated with an equal volume of denatur-ation solution for5min on ice.According to the manu-facturer’s protocol,RNA extraction was performed by acid-phenol:chloroform,and precipitation was carried out by ethanol and a?lter cartridge.The extracted RNA was eluted in100l L of preheated elution solution and mea-sured on a NanoDrop ND-1000Spectrophotometer (Thermo Scienti?c,Wilmington,USA).The RNA samples were immediately stored at-80°C and within few days converted into cDNA.

Conversion of total RNA into cDNA

A day before performing quantitative real-time PCR,the RNA was converted into cDNA.Reverse transcription was performed by the TaqMan MicroRNA Reverse Transcrip-tion Kit(Applied Biosystems,Darmstadt,Germany).The

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Table1Patient characteristics at the time of primary diagnosis of breast cancer and the relative serum levels miRs of patients with breast cancer,patients with benign breast disease and healthy controls

Parameters n(%)a miR-19a miR-20a miR-21miR-214

Mean Median Mean Median Mean Median Mean Median

95%CI b95%CI b95%CI b95%CI b

Patients with breast cancer

Total102(100)221210910429624153

15–2897–122251–3413–6

Age60years(range30–82)

Histology

Ductal64(63)191110210030024153

13–2587–116236–3643–7

Lubular21(21)251412510733525144

4–4688–161230–4402-6

Others16(16)31101159629222253

1–6381–149193–3902–8

Tumor stage

pT145(44)29141029730224153

15–4382–121227–3783–8

pT2-457(56)161111510528824243

11–2198–132232–3443–6

Lymph node metastasis

Neg.54(53)2812959527423642

15-4081–109227–3222–5

Pos.48(47)151212510731724564

11–19104–146*235–3993–9**

Grading

1,257(56)231110910533830142

14–3392–126270–4073–5

344(44)19121099923718963

10–2989–129186–2883–9

Tumor size

\2cm47(47)30131019630724163

15–4482–120233–3823–8

[2cm53(53)151111610628324243

11–1998–133227–3393–5

Estrogen receptor

Neg.20(20)231212211627120542

1–4483–162160–3812–5

Pos.82(80)221210610329924553

15–2993–119248–3503–7 Progesterone receptor

Neg.36(35)141110310630424163

9–1986–120209–3993–9

Pos.66(65)261211210228923942

16–3695–130241–3373–5

Recidive

No49(48)23111089629726043

13–3490–126242–3533–5

Yes52(52)211210710732224573

10–3290–125232–4114–10

10l L-reverse transcription reaction contained0.1l L100 mM dNTPs,0.66l L MultiScribe Reverse Transcriptase (50U/l L),1l L109Reverse Transcription Buffer, 0.13l L RNase Inhibitor(20U/l L),nuclease-free water and3.33l L or1l L RNA derived from human serum or cultured cells,respectively.On a MJ Research PTC-200 Peltier Thermal Cycler(Global Medical Instrumentation, Minnesota,USA)the reaction was carried out at16°C for 30min,42°C for30min,and85°C for5min. Quantitative real-time PCR of miR-19a,miR-20a,

miR-21and miR-214

For quantitative real-time PCR,the miR-speci?c TaqMan MicroRNA Assays(Applied Biosystems)for miR-16 (reference miR),miR-19a(locus:13q31.3),miR-20a (13q31.3),miR-21(17q23.1),and miR-214(1q24.3)were used.In a10l L-reaction,1l L cDNA was mixed with 5l L TaqMan Universal PCR Master Mix No AmpErase UNG and0.5l L miR-speci?c TaqMan MicroRNA Assay Mix on a twin-tec real-time PCR plate(Eppendorf,Ham-burg,Germany).The quantitative real-time PCR reaction was performed at95°C for10min and in40cycles at95°C for15s and60°C for60s on a Mastercycler Realplex (Eppendorf).Melting curve analyses were performed to verify the speci?city and identity of PCR products.

The obtained data of the miR expression levels were calculated and evaluated by the D Cq method as follows: D Cq=mean value Cq(reference miR-16)—mean value Cq(miR of interest).The relative expression of miR of interest corresponded to the2^(D Cq)value.As recom-mended by the manufacturer(Applied Biosystems),miR-16has been chosen as reference for normalization of the expression levels of our miR panel.To effectively nor-malize the expression data of miR-19a,miR-20a,miR-21, and miR-214,we analyzed the miR-16expression levels and found stable levels remained across the serum samples. We calculated a mean value of22.88,20.79,and22.00 with a standard deviation of2.28,0.99,and1.99for the groups of breast cancer patients,patients with benign breast disease and healthy women,respectively.Standardized RNA volumes were used for real-time PCR,resulting in similar mean values of miR-16in the patient cohort and healthy controls.

Statistical analyses

The statistical analyses were performed using the SPSS software package,version18.0(SPSS Inc.Chicago,IL). The v2or two-tailed Fischer0s exact test was used to identify potential associations between miR concentrations in blood serum and the clinical and histopathological risk factors of the breast cancer patients.For non parametric comparisons,univariate analyses of the Mann–Whitney U test of two independent variables and Wilcoxon test of two dependent variables were used.In addition,univariate binary logistical regression and bivariate analyses of the Spearman-Rho test were carried out.Diagnostic power of the single markers was analyzed by receiver operating characteristic(ROC)curves.Areas under the curves(AUC)

Table1continued

Parameters n(%)a miR-19a miR-20a miR-21miR-214

Mean Median Mean Median Mean Median Mean Median

95%CI b95%CI b95%CI b95%CI b

Dead

No20(20)231211210631426953

14–3199–125267–3613–7

Yes81(80)18111018530718063

6–3062–141113–5012–9

Patients with benign breast tumors

Total32(100)11101311123273370.70.6

8–14102–160247–4060.5–0.9

Age50years(range26–63)

Healthy women

Total53(100)1210766999960.50.3

10–1464–8782–1170.3–0.6

Age50years(range20–79)

a n number of patients or events

b CI con?dence intervals refers to the mean value

*p=0.041,**p=0.039,in bold

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were calculated.Missing data were handled by pairwise deletion.A p -value of \0.05was considered as statisti-cally signi?cant.All p -values are two-sided.

Results

Pro?ling of circulating miR-19a,miR-20a,miR-21,and miR-214in serum of patients with malignant and benign breast disease

In a quantitative RT-PCR we measured the relative expression levels of the miR transcripts by determining the low cycle threshold (Cq)values.In the box plot of Fig.1a,the relative quanti?cation of circulating miR-19a,miR-20a,miR-21,and miR-214in serum of 53healthy women,32patients with benign breast disease and 102patients with

early breast cancer are depicted.To determine the differ-ences in the relative expression pro?les (normalized Cq values),we performed univariate analyses of the Mann–Whitney U test.Table 1summarizes the mean and median values,as well as the 95%con?dence intervals (CI)of the miR variables in the cohorts of patients with breast cancer,patients with benign breast tumors and healthy women.As shown in Fig.1a,patients with breast cancer or benign breast disease had signi?cantly higher levels of circulating miR-20a and miR-21in their serum than healthy individ-uals (p =0.0001).Their mean and median values were approximately 1.5–3.5fold higher than the values detected in healthy women (Table 1).However,these high miR levels could not discriminate between benign and malig-nant disease.The levels of miR-19a were similar in the cohorts of healthy women and patients.In contrast,the concentrations of miR-214were signi?cantly higher in

p=0.0001p=0.0001p=0.0001p=0.0001p=0.0001

p=0.0001

b

miR214:AUC=0.878(95% CI: 0.818-0.937)

c

a

miR-20a:AUC=0.675(95% CI: 0.585-0.765)miR-21:AUC=0.845(95% CI: 0.784-0.907)miR-214:AUC=0.924(95% CI: 0.883-0.966)

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breast cancer patients than in serum of patients with benign disease and healthy women (p =0.0001),and thus,could discriminate between benign and malignant disease (Fig.1a).In breast cancer patients the mean and median values of miR-214were *5–10fold higher than the values detected in patients with benign breast disease and healthy women (Table 1).The AUC values of miR-20a,miR-21,and miR-214were 0.675,0.845,and 0.924,respectively,demonstrating the discriminatory power of the transcript levels to distinguish between breast cancer patients and healthy individuals (Fig.1b).Moreover,in Fig.1c the impressive strength of miR-214to discriminate between breast cancer patients and patients with benign disease is illustrated by the AUC value of 0.878.

Comparison of miR-19a,miR-20a,miR-21,

and miR-214levels in pre-and postoperative serum of breast cancer patients

Postoperative serum samples from 34breast cancer patients (3–8months after surgery)were available.To obtain information on the serum levels of our miR panel after surgery,an examination of these samples was per-formed.As shown in the box plot of Fig.2,only the levels of miR-214signi?cantly decreased after surgery (p =0.0001,Wilcoxon test),whereas the levels of the other miRs did not change signi?cantly.The cohort of 34patients was too small to analyze the potential association between their chemotherapy-induced changes in the serum yields of miRs and the patient response to chemotherapy.

Correlation of circulating miRs in serum of breast cancer patients with clinical and histopathological factors

We compared the concentrations of circulating miR-19a,miR-20a,miR-21,and miR-214in blood serum of the 102breast cancer patients with their clinical and histopatholo-gical data.For these statistical evaluations,the Mann–Whitney U test,binary logistical regression and Log rank test were used.Table 1summarizes the mean and median values,as well as the 95%CI of the miR variables in the different patient subgroups.MiR-20a (p =0.041)and miR-214(p =0.039)were associated with lymph node metastases (Table 1,in bold).The combination of miR-20a with miR-21correlated with tumor grading of the breast cancer patients (p =0.044).

Discussion

In the current study we quanti?ed circulating cell-free miR-19a,miR-20a,miR-21,and miR-214,which have been reported to target 30-UTR of PTEN mRNA,in serum of patients with breast cancer and benign breast disease as well as healthy women.To date,no studies on circulating miRs in blood of patients with benign breast disease have been published.Considering the different miR amounts in the serum,the levels of miR-19a were similar in the patients with malignant and benign disease and healthy women,and thus,miR-19a had no diagnostic relevance in our study.Moreover,the levels of miR-20a and miR-21were signi?cantly elevated in both patient groups as com-pared to healthy women.However,both miRs could not discriminate between benign and malignant breast tumors.The reasons for these high levels in the serum of patients with benign breast disease are unclear,but could be explained among others by the fact that malignant and benign lesions re?ect similar pathological and in?amma-tory processes.MiR-214was the only one those amounts were higher in breast cancer patients than in patients with benign disease and healthy women.As illustrated by the ROC curves with AUC values of *0.9,the difference of the levels of miR-214between breast cancer patients and patients with benign disease or healthy women was highly signi?cant.These ?ndings indicate that the concentrations of miR-214were cancer-speci?cally increased in the serum.

Moreover,the comparison of pre-and postoperative serum samples showed that only the levels of miR-214decreased after surgery,whereas the concentrations of the other miRs did not change in our study.This signi?cant decline of postoperative serum levels of miR-214might be a consequence of the resection of the tumors 3–8months

p=0.0001p=0.0001

p=0.0001

p=0.0001

p=0.0001

p=0.0001p=0.0001

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before taking these blood samples for our miR analysis. Thus,our?ndings suggest that miR-214might be released by the primary tumor.As the serum yields of miR-214did not associate with tumor size and stage,we cannot exclude that miR-214may also stem from other tissue sources,such as mononuclear blood cells,circulating tumor cells or occult micrometastases.However,a potential relationship of the serum yields of miR-214with these parameters could not be investigated,because the data on the number of leukocytes and CTCs were not collected.Also in?am-matory processes might be involved in this release,because the serum levels of miR-214in the cancer patients merely decreased after surgery to the corresponding levels detec-ted in patients with benign disease.Moreover,it can only be speculated why the levels of miR-20a and miR-21 remained elevated after surgery.Here,in?ammatory pro-cesses might especially contribute to these steady levels.In contrast to the preoperative concentrations of miR-214 which were signi?cantly higher than the serum concen-trations in patients with benign disease,the levels of miR-20a and miR-21were similar in preoperative cancer patients and patients with benign tumors.The postoperative levels of miR-214,miR-20a and miR-21which are,thus, similar to the corresponding levels in patients with benign disease,but higher than the corresponding levels in healthy women,were likely to be caused by in?ammatory lesions. Besides,also other sources might contribute to the release of these miRs into the blood circulation,such as circulating tumor cells or occult micrometastatic deposits being resistant to systemic treatment[20,21].

Our hitherto investigations demonstrate the cancer-spe-ci?c increase in levels of miR-214.In addition,the increased levels of this miR are also associated with positive lymph node status,indicating that miR-214might be involved in cancer progression.Thus,our?ndings suggest that quanti?cation of miR-214might be suitable for detecting breast cancer and lymph node metastases,and monitoring of residual tumor burden in patients before and after surgery.So far,no investigations of miR-214have been carried out in blood and tissue samples from breast cancer patients.In line with our data,it was reported that in gastric and colorectal cancer patients the plasma concen-trations of miR-214were signi?cantly reduced in postop-erative samples[22,23].In ovarian cancer miR-214may be involved in chemosensitivity,and knockdown of miR-214reduced cell survival by20%in cisplatin resistant ovarian cancer cells[15].In immortalized ovarian surface epithelial(HIOSE)cell lines[15],T cells[24],and monocytes[25]miR-214was reported to target PTEN and consequently,to increase cell proliferation and delay apoptosis.Since PTEN is an important target gene of miR-214in these cells,PTEN could also be a potential target of miR-214in breast cancer.This suggests that our?ndings could also have potential implications for therapeutic approaches of breast cancer patients.It was reported that in breast cancer patients treated with trastuzumab,an effec-tive agent for HER2-postive cancer patients,the loss of PTEN was associated with decreased survival of these patients[26].Therefore,we plan further studies to inves-tigate whether the downregulation of PTEN is associated with the upregulation of miR-214in these patients and whether miR-214is involved in the mechanism underlying the resistance to trastuzumab which numerous patients develop[27].To date,in vitro experiments employing established breast cancer cell lines and in vivo xenograft studies have demonstrated the ef?cacy of oligonucleotide-based overexpression and inhibitor approaches of miR-targeted experimental therapies[28].

Regarding the chromosomal miR loci,miRs are fre-quently located in fragile,cancer-associated genomic areas [29].The polycistronic cluster of miR-17-92at the locus 13q31,which also contains the genes encoding for miR-19a and miR-20a,has been reported to be among the most potent oncogenic miRs.Genomic ampli?cation and high transcript levels of miR17-92were detected in human B-cell lymphomas.In multiple mouse tumor models its elevated expression exhibited strong tumorigenic activity. MiR-17-92has been described to promote cell prolifera-tion,inhibit differentiation,increase angiogenesis,and sustain cell survival[30].Emerging evidence shows that the cluster may also have a role in breast cancer tumori-genesis.Expression of the miR-17-92cluster was higher in metastatic breast cancer cells than in non-metastatic cells [31].However,as far as we know,no investigations on miR-17-92have been performed in benign tumor tissues to examine whether miR-17-92discriminates between malignant and benign tumors.In our analyses,miR-19a had no diagnostic potential in discriminating these patient cohorts and even,could not differentiate between healthy and diseased individuals.Therefore,this miR seems to be less clinically relevant in our population.To date,there are no other studies on miR-19a in breast cancer patients.

Although in our study the levels of miR-20a were ele-vated in benign and malignant breast tumors,their dereg-ulated transcript levels correlated with positive lymph node status.It was reported,that increased concentrations of miR-20a were also observed in the plasma of patients with B-cell chronic lymphocytic leukemia and the levels cor-related with diagnosis-to-treatment time[32].Moreover, when we combined the elevated serum values of miR-20a with those of miR-21,these levels signi?cantly associated with tumor grading of the patients.Hence,our data indicate that the combined levels of miR-20a and miR-21correlated with cancer dedifferentiation.Apart from this correlation of the combination with miR-20a and the elevated levels in both patient cohorts,miR-21was not speci?c any more in

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our study.We only quanti?ed miR-21in serum samples from patients with early breast cancer.Therefore,we do not know whether patients with metastatic breast cancer have speci?cally increased serum levels of miR-21,but in a recent report high serum concentrations of circulating miR-21were detected in breast cancer patients with visceral metastasis[33].In breast cancer epithelium tissue over-expression of miR-21correlated with lymph node metas-tasis,emphasizing the role of miR-21in metastasis,but the population size used was relatively small[34].In addition, multivariate analyses identi?ed miR-21in bone marrow as a valuable independent prognostic factor based on the high levels of miR-21which signi?cantly associated with shortened disease-free survival.These?ndings suggested that miR-21may accumulate in bone marrow[35].In patients with esophageal squamous cell carcinoma high plasma levels of miR-21tended to have greater vascular invasion and correlated with recurrence[36].For the early diagnosis of non-small cell lung cancer(NSCLC)the increased plasma levels of miR-21could serve as a circu-lating tumor biomarker and was related to the sensitivity to platinum-base chemotherapy[37].In serum of patients with diffuse large B-cell lymphoma high miR-21levels were associated with relapse-free survival[17].

In conclusion,the key?nding of our study is that serum levels of miR-214discriminate between benign and malignant breast tumors,and correlate with tumor burden and lymph node metastases.To further validate the clinical utility of circulating miR-214,a prospective large-scale study is planned.In addition,functional analyses are intended,to characterize the cancer-speci?c increase in its serum concentrations and screen for further targets of miR-214.

Acknowledgment We are grateful to the European Research Council(Advanced Investigators Grant269081-DISSECT)and the Erich und Gertrud Roggenbuck-Stiftung,Hamburg,for supporting this study.We thank Ms.Carina Roth for their excellent technical assistance.

Con?ict of interest All authors disclose any?nancial and personal relationships with other people or organizations that could inappro-priately in?uence(bias)their study.

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