2004 MicroRNA profiling reveals distinct signatures in

MicroRNA profiling reveals distinct signatures in

B cell chronic lymphocytic leukemias

George Adrian Calin*?,Chang-Gong Liu*?,Cinzia Sevignani*,Manuela Ferracin?,Nadia Felli*§,Calin Dan Dumitru*, Masayoshi Shimizu*,Amelia Cimmino*,Simona Zupo?,Mariella Dono?,Marie L.Dell’Aquila**,Hansjuerg Alder*, Laura Rassenti**??,Thomas J.Kipps**??,Florencia Bullrich*??,Massimo Negrini*?,and Carlo M.Croce*????

*Kimmel Cancer Center,Thomas Jefferson University,Philadelphia,PA19107;?Department of Experimental and Diagnostic Medicine and Interdepartment Center for Cancer Research,University of Ferrara,44700Ferrara,Italy;§Department of Hematology,Oncology,and Molecular Medicine,Istituto Superiore Sanita,00161Rome,Italy;?Diagnostics of Lymphoproliferative Diseases,National Institute of Cancer,16123Genoa,Italy;?Laboratorio di Analisi Cliniche, Ospedale Civile La Spezia,19126La Spezia,Italy;**Department of Medicine,University of California at San Diego,La Jolla,CA92093;and??Chronic Lymphocytic Leukemia Research Consortium,University of California at San Diego,La Jolla,CA92093-0675

Contributed by Carlo M.Croce,June21,2004

Little is known about the expression levels or function of micro-RNAs(miRNAs)in normal and neoplastic cells,although it is becoming clear that miRNAs play important roles in the regula-tion of gene expression during development[Ambros,V.(2003) Cell113,673–676;McManus,M.T.(2003)Semin.Cancer Biol.13, 253–258].We now report the genomewide expression pro?ling of miRNAs in human B cell chronic lymphocytic leukemia(CLL)by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes.This approach allowed us to identify signi?cant differences in miRNome expression between CLL samples and normal CD5?B cells;data were con?rmed by Northern blot analyses and real-time RT-PCR.At least two distinct clusters of CLL samples can be identi?ed that were associated with the presence or absence of Zap-70expression,a predictor of early disease progression.Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variable-region genes or with deletions at13q14,respectively.These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia.

M icroRNAs(miRNAs)represent a class of small,func-tional,noncoding RNAs of19–23nt cleaved from?60-to 110-nt hairpin precursors(1,2).Hundreds of miRNAs have been identified in plants and animals.The miRNAs are involved in various biological processes,including cell proliferation and cell death during development,stress resistance,and fat metabolism, through the regulation of gene expression(3).Some miRNAs, such as miR-15a or miR-16–1(4,5),are widely expressed, whereas others,such as miR-1in mammalian heart(6,7)or miR-223in granulocytes and macrophages(5),are expressed in a tissue-specific manner.Little else is known about miRNA expression patterns or function in normal or neoplastic cells. Understanding of the molecular pathogenesis of B cell chronic lymphocytic leukemia(CLL),the most common adult leukemia in the Western world,is incomplete.We have shown previously that miR-15a and miR-16-1are located at chromosome13q14.3 within a30-kb region of loss in CLL cells and that both genes are deleted and?or down-regulated in the majority of the analyzed CLL cell samples(4).These results provided the indication that deletion of miRNAs might be associated with a human malig-nancy.We also reported that98of the identified186miRNAs are located at fragile sites,minimal loss of heterozygosity regions,minimal regions of amplification,or common break-point regions in human cancers(8),suggesting that miRNAs might play a large and unanticipated role in the pathogenesis of human cancer.

Methods

Tissue Samples and CLL Samples.Forty-seven samples were used for this study,including41samples from38patients with CLL and 6normal samples,including one lymph node,tonsillar CD5?B cells from two normal donors,and blood mononuclear cells (MNC)from three normal donors.For three cases,two inde-pendent samples were collected and processed.CLL samples were obtained after informed consent from patients diagnosed with CLL at the CLL Research Consortium institutions.Briefly, blood was obtained from CLL patients,and MNC were isolated through Ficoll?Hypaque gradient centrifugation(Amersham Pharmacia Biotech)and processed for RNA extraction accord-ing to described protocols(9).For the majority of samples, clinical and biological information,such as age at diagnosis,sex, Rai stage,presence?absence of treatment,ZAP-70expression, and IgV H gene mutation status were available(see Table4, which is published as supporting information on the PNAS web site).

Cell Preparation.MNC from peripheral blood of normal donors were separated by Ficoll-Hypaque density gradients.T cells were purified from these MNC by rosetting with neuraminidase-treated sheep erythrocyte and depletion of contaminant mono-cytes(Cd11b?);natural killer cells(CD16?)and B lymphocytes (CD19?)were purified by using magnetic beads(Dynabeads, Unipath,Milan)and specific mAbs(Becton Dickinson).Total B cells and CD5?B cells were prepared from tonsillar lympho-cytes as described(10).Briefly,tonsils were obtained from patients in the pediatric age group undergoing routine tonsil-lectomies,after informed consent.Purified B cells were pre-pared by rosetting T cells from MNC with neuraminidase-treated sheep erythrocyte.To obtain CD5?B cells,purified B cells were incubated with anti-CD5mab followed by goat anti-mouse Ig conjugated with magnetic microbeads.CD5?B cells were positively selected by collecting the cells retained on the magnetic column MS by Mini MACS system(Miltenyi Biotec,Auburn,CA).The degree of purification of the cell preparations was?95%,as assessed by flow cytometry.

RNA Extraction and Northern Blots.Total RNA isolation and blots were performed as described(4).After RNA isolation,the washing step with ethanol was not performed or if performed, the tube walls were rinsed with75%ethanol without perturbing the RNA pellet(9).For reuse,blots were stripped by boiling in 0.1%aqueous SDS?0.1?SSC for10min and reprobed.5S rRNA stained with ethidium bromide served as a sample loading control.

Microarray Experiments.RNA blot analysis was performed as described(11).Briefly,labeled targets from5?g of total RNA

Abbreviations:miRNA,microRNA;CLL,chronic lymphocytic leukemia;MNC,mononuclear cells.

?G.A.C.and C.-G.L.contributed equally to this work.

??To whom correspondence should be addressed.E-mail:c?croce@https://www.sodocs.net/doc/6b15424020.html,.

?2004by The National Academy of Sciences of the USA

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was used for hybridization on each KCC ?TJU miRNA microar-ray chip containing 368probes in triplicate,corresponding to 245human and mouse miRNA genes.All probes on these microar-rays are 40-mer oligonucleotides spotted by contacting technol-ogies and covalently attached to a polymeric matrix.The mi-croarrays were hybridized in 6?SSPE (0.9M NaCl ?60mM NaH 2PO 4?H 2O ?8mM EDTA,pH 7.4)?30%formamide at 25°C for 18h,washed in 0.75?TNT (Tris ?HCl ?NaCl ?Tween 20)at 37°C for 40min,and processed by using a method of direct detection of the biotin-containing transcripts by streptavidin-Alexa647conjugate.Processed slides were scanned by using a PerkinElmer ScanArray XL5K Scanner,with the laser set to 635nm,at Power 80and PMT 70setting,and a scan resolution of 10?m.

Data Analysis.Expression profiles were analyzed in duplicate

independent experiments starting for three different patients.Raw data were normalized and analyzed in GENESPRING soft-ware version 6.1.1(Silicon Genetics,Redwood City,CA).GENE -SPRING generated an average value of the three spot replicates of each miRNA.After data transformation (to convert any negative value to 0.01),normalization was performed by using a per-chip on median normalization method and a normalization to specific samples,expressly to the two CD5?B cell samples,used as common reference for miRNA expression.Hierarchical clustering was generated,for both genes and conditions,by using standard correlation as a measure of similarity.To identify genes with statistically significant differences between sample groups (i.e.,CLL cells and CD5?B cells,CLL and MNC,CLL samples with or without IgV H mutations,or CLL cases with or without 13q14.3deletion),a Welch ’s approximate t test for two groups (variances not assumed equal)with a P value cutoff of 0.05and Benjamini and Hochberg false discovery rate as multiple testing correction were performed.

Real-Time PCR.Quantitative real-time PCR was performed as

described (12).Briefly,RNA was reverse-transcribed to cDNA with gene-specific primers and Thermoscript,and the relative amount of each miRNA to tRNA for initiator methionine was described,using the equation 2-dC T ,where dC T ?(C TmiRNA ?C TU6or HUMTMI RNA ).The set of analyzed miRNAs included miR-15a ,miR-16-1,miR-18,miR-20,and miR-21.The primers used were as published (12).

Western Blotting.Protein lysates were prepared from the leuke-

mia cells of seven CLL patients and isolated tonsillar CD5?B cells.Western blot was performed with a polyclonal Pten antibody (Cell Signaling Technology,Beverly,MA)and was normalized by using an anti-actin antibody (Sigma).

Microarray Data Submission.All data were submitted by using

MIAMEXPRESS

to the Array Express database,and each of the 38

CLL samples described here received an ID number ranging from SAMPLE169194SUB621to SAMPLE169234SIUB621.

Results and Discussion

Distinct miRNome Expression Signatures in Various Human Tissues Revealed with an Oligonucleotide Microchip.To investigate expres-

sion of each member of this class of genes in normal and neoplastic cells,we developed a microarray chip that can exam-ine global expression levels for each known member of the miRNome (defined as the full cellular complement of miRNA)(11).This chip contains gene-specific 40-mer oligonucleotide probes generated from 161human and 84mouse precursor miRNA collected from the Sanger database (July 2003release)or from published papers (9,13,14).In some instances,two different oligonucleotide probes were designed,one containing

the active sequence and the other specific for the precursor,enabling us to analyze expression of the mature miRNA and its precursor at the same time.We tested this platform by using a panel of 20human normal tissues and identified specific miRNA expression signatures for each tissue type (11).Based on these signatures,hematopoietic tissues cluster together in a group distinct from that of nonhematopoietic tissues,showing that miRNA expression profiles differ with cell ?tissue type,suggest-ing that abnormal cell ?tissues will also have distinctive miRNA expression profiles.

B-CLL Cells Present Distinct miRNome Signatures in Respect with CD5?Cells.To further investigate the involvement of miRNA in

CLL,we analyzed the miRNome expression in 38individual CLL cell samples.One normal lymph node sample and five samples from healthy donors,including two tonsillar CD5?B lympho-cyte samples and three blood MNC samples,were included for comparison.Unsupervised hierarchical clustering revealed ho-mogenous but distinctive expression profiles for each sample type,with all CLL samples falling into a cluster that was distinct from that of the lymph node or normal lymphoid cell samples (Fig.1).First,we compared miRNA expression in CLL cells vs.normal CD5?B cells,considered as a normal cell counterpart to the CLL B cells,or CLL cells vs.normal blood MNC.As expected,two groups of differentially expressed miRNAs,the first composed of 55genes and the second of 29genes,had statistically significant differences in expression levels between the various groups (P ?0.05using the Welch t test as described in Methods )(Table 1and Table 5,which is published as supporting information on the PNAS web site).Of note,only six miRNAs are shared between the two lists,a result

confirming

Fig.1.Two distinct miRNA signatures characterize the CLL samples.Thirty-eight CLL patients were analyzed;from three of them two different cell samples were processed and analyzed.In all instances,the different samples from the same patients clustered together.The main miRNA-associated CLL clusters are presented.The control samples are:MNC;Ly,lymph node;CD5?,selected CD5?B lymphocytes.

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our previous data showing distinct miRNome signatures in CD5?B cells and leucocytes(11).When both pre-miRNA and mature miRNA were observed to be dysregulated(such as for miR-123,miR-132,or miR-136),the same type of variation in CLL samples with respect to CD5or MNC was noted in every case.Also,for some miRNA genomic clusters all members were aberrantly regulated(such as the up-regulated7q32group encompassing miR-96–miR182–miR183),whereas for others only some members were abnormally expressed(such as the 13q31genomic cluster where two of six members,miR-19and miR-92-1,were strongly up-regulated and two,miR-17and miR-20,were moderately down-regulated)(Tables1and5).The results illustrate the complexity of the patterns of miRNA expression in CLL and argue for the existence of mechanisms regulating individual miRNA genes that map in the same chromosome region.In confirmation of the data accuracy, miR-223,reported to be expressed at high levels in granulocytes (5),was expressed at significantly lower levels in the CLL samples than in the MNC,but at about the same level as that noted for CD5?B cells(which generally constitute less than a few percent of blood MNC).

At the top of the CLL vs.CD5?B cell list are several miRNAs located exactly inside fragile sites(miR-183at FRA7H,miR-190at FRA12A,and miR-24-1at FRA9D)and miR-213.The mature miR-213molecule is expressed at lower levels in all of the CLL samples,and the precursor miR-213is reduced in expression in 62.5%of the samples.miR-16-1,at13q14.3,which we previously reported to be down-regulated in the majority of CLL cases by Northern analysis(4),was expressed at low levels in45%of CLL samples.An identical mature miR-16exists on chromosome3; because the40-mer oligonucleotide for both miR-16sequences from chromosome13(miR-16-1)and chromosome3(miR-16-2) exhibit the same23-mer mature sequence,very similar profiles were observed.However,because we observed very low levels of miR-16-2expression in CLL samples by Northern blot,the expression observed is contributed mainly by miR-16-1.The other miRNA of 13q14.3,miR-15a,was expressed at low levels in?25%of CLL cases.Overall,these data demonstrate that CLL is a malignancy with extensive alterations of miRNA expression and suggest a role for distinct miRNAs in the pathogenesis of human B cell malig-nancies(Fig.1and Fig.3,which is published as supporting information on the PNAS web site).

Validation of the microarray data was supplied for four miRNAs by Northern blot analyses:miR-16-1,located within the region of deletion at13q14.3,miR-26a,on chromosome3in a region not involved in the pathogeneses of CLL,and miR-206and miR-223 that are down-regulated(see above)in the majority of samples.For all four miRNAs,the Northern blot analyses confirmed the data obtained by using the microarray(Fig.2A).We also performed real-time RT-PCR to measure expression levels of precursor mol-ecules for five genes(miR-15a,miR-16–1,miR-18,miR-21,and miR-30d),and we found results concordant with the chip data (examples for miR-15a and miR-30d are presented in Fig.2B).

miRNA Signatures in B-CLL Cells Are Associated with the Levels of ZAP-70.Second,we asked whether CLL cells from different patients shared the same miRNA expression patterns.Unsuper-vised hierarchical clustering generated two clearly distinguish-able miRNA signatures within the set of CLL samples,one closer to the miRNA expression profile observed in human MNC and the other clearly different(Fig.1).The top25differentially expressed miRNA in these two signatures(at P?0.001)include genes known or suggested to be involved in cancer(Table2and Table6,which is published as supporting information on the PNAS web site).The precursor of miR-155is overexpressed in the majority of childhood Burkitt’s lymphoma(15),miR-21is located at the fragile site FRA17B(8),and miR-26a is at3p21.3, a region frequently deleted region in epithelial cancers,whereas miR-92-1and miR-17are at13q32,a region amplified in malignant lymphomas(16).Furthermore,we asked whether there are some clinico-biological factors that can help to dis-criminate between the two clusters.We found a high difference in the levels of ZAP-70between the two groups:66%(6?9) patients from the first cluster vs.25%(4?16)patients from the second one(Fig.1)have low levels of ZAP-70(?20%)(P?0.04 at?test).The mean value of ZAP-70was19%(?31%SD)vs. 35%(?30%SD),respectively;otherwise the two clusters can discriminate between patients who express and who do not express this protein(at levels?20%ZAP-70is considered as nonexpressed)(Table4).ZAP-70is a tyrosine kinase,which is a strong predictor of early disease progression,and low levels of expression are proved to be a finding associated with good prognosis(17).To validate these important differences further studies with larger numbers of patients are required.

Chromosome13q14Deletions and the Expression of Mutated IgV H Correlates with miRNAs Expression.Third,we asked whether the microarray data revealed specific molecular signatures predic-tive for subsets of CLL that differ in clinical behavior.CLL cases harbor deletions at chromosome13q14.3in?50%of cases(18). As a single cytogenetic defect,these CLL patients have a relatively good prognosis,compared with patients with leukemia cells harboring complex cytogenetic changes(19).It was also shown that deletion at13q14.3was associated with the presence of mutated IgV H genes(20),another good prognostic factor.By comparing expression data of CLL samples with or without deletions at13q14,we found that miR-16-1was expressed at low levels in leukemias harboring deletions at13q14(P?0.03, ANOVA).We also found that miR-24-2,miR-195,miR-203, miR-220,and miR-221are expressed at significantly reduced levels,whereas miR-7-1,miR-19a,miR-136,miR-154,miR-217,

Table1.Top25miRNAs differentially expressed in CLL cells versus CD5?cells

miRNA Chromosome

location

FRA

associated P value Type

mir-2131q31.3–q32.1 1.47E-33Down mir-183-prec7q32FRA7H 1.26E-23Up mir-19015q21FRA15A 1.48E-20Up mir-24-1-prec9q22.1FRA9D7.35E-20Up mir-3322q13.2 1.56E-18Up mir-19a13q31 5.16E-17Up mir-14016q22.1 2.41E-16Up mir-1239q34 2.80E-16Up mir-10b2q31 1.10E-15Up mir-15b-prec3q26.1 5.79E-14Up mir-92-113q31 1.70E-12Up mir-188Xp11.23-p11.2 6.08E-11Up mir-15414q32 1.14E-10Up mir-220Xq25 2.14E-09Down mir-2172p16 3.85E-09Up mir-1011p31.3FRA1C 1.26E-08Up mir-141-prec12p13 1.39E-08Up mir-153-prec2q36 1.48E-08Up mir-196-212q13FRA12A 4.94E-08Up mir-13414q32 6.01E-08Up mir-14112p137.91E-08Up mir-13211q12 1.68E-07Up mir-19211q13 2.00E-07Down mir-181b-prec1q31.2-q32.1 3.26E-06Up For a complete list see Table5.The name of each miRNA is as in the miRNA Registry,and the disregulation of either active molecule or precursor is speci?ed in the name.The type of altered expression is presented in the last column.

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and the precursor of miR-218-2are expressed at significantly higher levels in the samples with 13q14.3deletions,respectively (Table 3).All of these genes are located in different regions of the genome and differ in their nucleotide sequences,excluding the possibility of cross-hybridization,suggesting the existence of functional miRNA networks in which hierarchical regulation may be present,with some miRNA (such as miR-16-1)control-ling or influencing the expression of other miRNA.

We also looked for a specific signature profile associated with the expression of mutated IgV H ,a favorable prognostic marker (20).Indeed,a distinct miRNA signature composed of five differentially expressed genes (miR-186,miR-132,miR-16-1,miR-102,and miR-29c )distinguished CLL samples that expressed mutated IgV H gene from those that expressed unmutated IgV H genes (Table 3),suggesting that miRNA expression profiles have prognostic signif-icance in CLL.A confirmation of our results is the observation that the common element between the del 13q14.3-related and the IgV H -related signatures is miR-16-1.This gene is located in the common deleted region 13q14.3and the presence of this particu-lar deletion is associated with good prognosis.Therefore,miRNAs may expand the spectrum of adverse prognostic markers in CLL,such as expression of ZAP-70,unmutated IgV H ,CD38,deletion at chromosome 11q23,or loss or mutation of TP53.

PTEN Protein Expression Inversely Correlates with the Levels of miR19Expression on the Microchip.A present limitation in our under-

standing of the function of miRNA is our lack of knowledge about specific targets.However,two recent publications have presented algorithms with which to identify putative targets for

miRNA in human (21)or Drosophila (22).Also,it might be possible to relate genomewide miRNA expression with data obtained from analyses by using large EST microarrays to identify miRNA gene targets,thereby revealing the functional consequences of altered miRNA gene expression in physiolog-ical or pathological states.We selected miR-19a ,because its targets were experimentally determined (21),and we found it as a significantly up-regulated miRNA in CLL cells in respect to CD5?B cells (Tables 3and 5).One of its targets is PTEN (21),a tumor suppressor gene expressed in both CD5?and CLL cells.It was reported that Pten protein is absent in CLL with a normal PTEN genotype,suggesting down-regulation of protein synthesis,only partly explained by promoter methylation (23).Analyzing a limited set of CLL cases,we have found an inverse correlation between the levels of expression of miR-19a and Pten protein in six of seven samples (86%),a result not observed for Mecp2protein (methyl-CpG-binding protein),a predicted target but not experimentally confirmed (24)(Fig.2C ).Thus,using the combination of Western blot and our microarray platform,it is possible to analyze the relation between expression of miRNA and putative targets in large sets of clinical samples.

Genomic expression profiling using microarrays has proven to be a powerful approach for relating gene expression profiles with cell signaling in normal and disease states (25).We have developed a microarray chip for the analysis of expression of hundreds of miRNAs.Despite the continuous expansion of the number of known miRNA in various organisms,the miRNA microchip ap-pears to be a powerful tool for analyzing miRNA expression patterns and quantitative expression level assays.This is

important

Fig.2.Microarray data con ?rmation.(A )Northern blots of CLL RNAs showing concordance with the microarray https://www.sodocs.net/doc/6b15424020.html,s of miRNA and speci ?c oligonucleotides spotted on the array are presented.The numbers correspond to absolute expression value of each miRNA (determined by a per-chip on median normalization)and,therefore can be compared with the band intensity on Northern blots.5S RNA stained with ethydium bromide (Et-Br)was used as loading control.The signi ?cance of the two oligos designed for some miRNAs is described in Tables 4–6.Generally,the oligoNo1corresponds to the active part,whereas the oligoNo2corresponds to the precursor miR molecule.PBL,peripheral blood leukocytes.(B )Real-time PCR quanti ?cation of miRNA expression in MNC and CLL samples.Expression of miR-15a (Left )and miR-30d (Right )by microarray (yellow bar)is compared with the levels detected with the two oligo speci ?c for the active part (red bar)or precursor miRNA (blue bar).Correlations only with the expression levels of pre-miRNA (blue bar)but not active miRNA (red bar)were found.The data represent the mean of triplicate real-time PCRs from a single DNA sample.(C )Variations in the expression of Pten protein,a target of miR-19a are paralleled,in six of seven CLLs,by miR-19a microarray expression.The numbers represent MeCP2,the methyl-CpG-binding protein excluded as a putative target of miR-19a was used as the negative control.The numbers correspond to absolute expression value of miR-19a (determined by a per-chip on median normalization).Actin was used as loading control.

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in light of the reported variations in expression levels of several miRNAs:reduced expression of miR-15a and miR-16-1in CLL(4), miR-143and miR-145in colon cancers(26),and let-7in lung cancers (27),or amplification of the precursor of miR-155in Burkitt lymphomas(15),and the13q31-q32miRNA cluster(miR-17, miR-91,miR-18,miR-19a,miR-20,miR-19b,and miR-92)in malig-nant lymphomas(16).By applying the miRNA microarray to the study of human CLL,we demonstrated that?200miRNA genes could reveal distinct miRNA signatures associated with specific bio-pathological features,including factors related to prognosis. The results suggest that miRNA plays important roles in the pathogenesis of human CLL.

Table2.miRNAs differentially expressed between the two main CLL clusters

miRNA Chromosome

location P value Cancer-associated genomic regions

miR-17-prec13q310.00000000Amp-folicular Ly?Del-HCC

miR-2013q310.00000000Amp-folicular Ly?Del-HCC

miR-103-220p130.00000001

miR-30d-prec8q24.20.00000002

miR-106Xq26.20.00000006Del-advanced ovarian cancer

miR-26b2q350.00000006

miR-103-15q35.10.00000006

miR-257q220.00000007FRA7F

miR-30a6q12–130.00000008

miR-2117q23.20.00000008Amp-neuroblastoma;FRA17B

miR-10710q23.310.00000008

miR-92-1-prec13q310.00000024Amp-folicular Ly?Del-HCC

miR-27a19p13.20.00000024

miR-23a19p13.20.00000032

miR-92-2Xq26.20.00000040Del-advanced ovarian cancer

miR-30b8q24.20.000004

miR-26a3p210.000009Del-epithelial malignancies

miR-93-17q220.000009Amp-folicular Ly?Del-HCC;FRA7F

miR-1941q410.000015FRA1H

miR-15521q210.000028Amp-colon cancer;childhood Burkit Lymphoma miR-153-27q360.000028t(7;12)(q36;p13)-AML

miR-193-prec17q11.20.000044Del-Muci?Sero ovarian cancer

miR-130a11q120.0001

miR-23b9q22.10.0001Del-urothelial cancer;FRA9D

miR-30c6q130.0001

miR-13911q130.0001

For a complete list see Table6.The name of each miRNA is as in the miRNA Registry,and the disregulation of either active molecule or precursor is speci?ed in the name.The location in minimally deleted or minimally ampli?ed or breakpoint regions or in fragile sites is presented.HCC,hepatocellular carcinoma;AML,acute myeloid leukemia;FRA,fragile site.

Table3.miRNA signatures associated with prognosis in B-CLL

miRNA Chromosome

location P value Association Observation

miR-7-19q21.330.03013q14normal

miR-16-113q14.30.030IGVH mutations negative

0.02313q14deleted

miR-19a13q310.02413q14normal

miR-24-219p13.20.03313q14deleted

miR-29c1q32.2–32.30.018IGVH mutations positive Cluster miR-29c-miR102 miR-1021q32.2–32.30.023IGVH mutations positive Cluster miR-29c-miR102 miR-13217p13.30.033IGVH mutations negative

miR-13614q320.04513q14normal

miR-15414q320.02013q14normal

miR-1861p310.038IGVH mutations negative

miR-19517p130.03613q14deleted

miR-20314q32.330.02613q14deleted

miR-217-prec2p160.00513q14normal

miR-218-25q35.10.01913q14normal

miR-220Xq250.02613q14deleted

miR-221Xp11.30.02113q14deleted

The name of each miRNA is as in the miRNA Registry,and the disregulation of either active molecule or

precursor is speci?ed in the name.IGVH,IgV H genes.

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This work was supported by National Institutes of Health Grant P01-CA81534(to the CLL Research Consortium),National Cancer Institute Grant CA076259,the Italian Ministero dell’Istruzione,dell’Universitae della Ricerca,and the Italian Ministero della Salute.

1.Moss,E.G.(2003)in MicroRNAs in Noncoding RNAs:Molecular Biology and

Molecular Medicine),eds.Barciszewski,J.&Erdmann,V.(Landes Bioscience, Georgetown,TX),pp.98–114.

2.Bartel,D.P.(2004)Cell116,281–297.

3.Ambros,V.(2003)Cell113,673–676.

4.Calin,G.A.,Dumitru,C.D.,Shimizu,M.,Bichi,R.,Zupo,S.,Noch,E.,Aldler,

H.,Rattan,S.,Keating,M.,Rai,K.,et al.(2002)https://www.sodocs.net/doc/6b15424020.html,A99,

15524–15529.

5.Chen,C.Z.,Li,L.,Lodish,H.F.&Bartel,D.P.(2004)Science303,83–8

6.

6.Lee,R.C.&Ambros,V.(2001)Science294,862–864.

https://www.sodocs.net/doc/6b15424020.html,gos-Quintana,M.,Rauhut,R.,Yalcin,A.,Meyer,J.,Lendeckel,W.&

Tuschl,T.(2002)Curr.Biol.12,735–739.

8.Calin,G.A.,Sevignani,C.,Dumitru,C.D.,Hyslop,T.,Noch,E.,Yendamuri,

S.,Shimizu,M.,Rattan,S.,Bullrich,F.,Negrini,M.&Croce,C.M.(2004)Proc.

https://www.sodocs.net/doc/6b15424020.html,A101,2999–3004.

https://www.sodocs.net/doc/6b15424020.html,gos-Quintana,M.,Rauhut,R.,Lendeckel,W.&Tuschl,T.(2001)Science

294,853–858.

10.Dono,M.,Zupo,S.,Leanza,N.,Melioli,G.,Fogli,M.,Melagrana,A.,

Chiorazzi,N.&Ferrarini,M.(2000)J.Immunol.164,5596–5604.

11.Liu,C.-G.,Calin,G.A.,Meloon,B.,Gamliel,N.,Sevignani,C.,Ferracin,M.,

Dumitru,D.C.,Shimizu,M.,Zupo,S.,Dono,M.,et al.(2004)Proc.Natl.Acad.

https://www.sodocs.net/doc/6b15424020.html,A101,9740–9744.

12.Schmittgen,T.D.,Jiang,J.,Liu,Q.&Yang,L.(2004)Nucleic Acid Res.32,

43–53.

13.Mourelatos,Z.,Dostie,J.,Paushkin,S.,Sharma,A.,Charroux,B.,Abel,L.,

Rappsilber,J.,Mann,M.&Dreyfuss,G.(2002)Genes Dev.16,720–728.

14.Lim,L.P.,Glasner,M.E.,Yekta,S.,Burge,C.B.&Bartel,D.P.(2003)Science

299,1540.15.Metzler,M.,Wilda,M.,Busch,K.,Viehmann,S.&Borkhardt,A.(2004)Genes

Chromosomes Cancer39,167–169.

16.Ota,A.,Tagawa,H.,Karnan,S.,Tsuzuki,S.,Karpas,A.,Kira,S.,Yoshida,Y.

&Seto,M.(2004)Cancer Res.64,3087–3095.

17.Orchard,J.A.,Ibbotson,R.E.,Davis,Z.A.,Wiestner,A.,Rosenwald,A.,

Thomas,P.W.,Hamblin,T.J.,Staudt,L.M.&Oscier,D.G.(2004)Lancet 363,105–111.

18.Bullrich,F.&Croce,C.M.(2001)Chronic Lymphoid Leukemia(Dekker,New

York).

19.Dohner,H.,Stilgenbaue,S.,Benner,A.,Leupolt,E.,Krober,A.,Bullinger,L.,

Dohner,K.,Bentz,M.&Lichter,P.(2000)N.Engl.J.Med.343,1910–1916.

20.Oscier,D.G.,Gardiner,A.,Mould,S.J.,Glide,S.,Davis,Z.A.,Ibbotson,R.E.,

Corcoran,M.M.,Chapman,R.M.,Thomas,P.W.,Copplestone,J.A.,et al.

(2002)Blood100,1177–1184.

21.Lewis,B.P.,Shih,I.H.,Jones-Rhoades,M.W.,Bartel,D.P.&Burge,C.B.

(2003)Cell115,787–798.

22.Rajewsky,N.&Socci,N.D.(2004)Dev.Biol.267,529–535.

23.Soria,J.C.,Lee,H.Y.,Lee,J.I.,Wang,L.,Issa,J.P.,Kemp,B.L.,Liu,D.D.,

Kurie,J.M.,Mao,L.&Khuri,F.R.(2002)Clin.Cancer Res.8,1178–1184.

24.Muller,H.M.,Fiegl,H.,Goebel,G.,Hubalek,M.M.,Widschwendter,A.,

Muller-Holzner,E.,Marth,C.&Widschwendter,M.(2003)Br.J.Cancer.89, 1934–1939.

25.Butte,A.(2002)Nat.Rev.1,951–960.

26.Michael,M.Z.,O’Connor,S.M.,van Holst Pellekaan,N.G.,Young,G.P.&

James,R.J.(2003)Mol.Cancer Res.1,882–891.

27.Takamizawa,J.,Konishi,H.,Yanagisawa,K.,Tomida,S.,Osada,H.,Endoh,

H.,Harano,T.,Yatabe,Y.,Nagino,M.,Nimura,Y.,et al.(2004)Cancer Res.

64,3753–3756.

11760?https://www.sodocs.net/doc/6b15424020.html,?cgi?doi?10.1073?pnas.0404432101Calin et al.