MicroRNA-218 Is Deleted and Downregulated in Lung Squamous Cell Carcinoma

MicroRNA-218Is Deleted and Downregulated in Lung Squamous Cell Carcinoma

Morgan R.Davidson1,2*,Jill https://www.wendangku.net/doc/4410873645.html,rsen1,2,Ian A.Yang1,2,Nicholas K.Hayward3,Belinda E.Clarke2,4, Edwina E.Duhig4,Linda H.Passmore1,Rayleen V.Bowman1,2,Kwun M.Fong1,2

1Department of Thoracic Medicine,The Prince Charles Hospital,Brisbane,Queensland,Australia,2Discipline of Medicine,University of Queensland,Brisbane, Queensland,Australia,3Oncogenomics Laboratory,Queensland Institute of Medical Research,Brisbane,Queensland,Australia,4Department of Anatomical Pathology, The Prince Charles Hospital,Brisbane,Queensland,Australia

Abstract

MicroRNAs(miRNAs)are a family of small,non-coding RNA species functioning as negative regulators of multiple target genes including tumour suppressor genes and oncogenes.Many miRNA gene loci are located within cancer-associated genomic regions.To identify potential new amplified oncogenic and/or deleted tumour suppressing miRNAs in lung cancer, we inferred miRNA gene dosage from high dimensional arrayCGH data.From miRBase v9.0(https://www.wendangku.net/doc/4410873645.html,), 474human miRNA genes were physically mapped to regions of chromosomal loss or gain identified from a high-resolution genome-wide arrayCGH study of132primary non-small cell lung cancers(NSCLCs)(a training set of60squamous cell carcinomas and72adenocarcinomas).MiRNAs were selected as candidates if their immediately flanking probes or host gene were deleted or amplified in at least25%of primary tumours using both Analysis of Copy Errors algorithm and fold change($61.2)https://www.wendangku.net/doc/4410873645.html,ing these criteria,97miRNAs mapped to regions of aberrant copy number.Analysis of three independent published lung cancer arrayCGH datasets confirmed that22of these miRNA loci showed directionally concordant copy number variation.MiR-218,encoded on4p15.31and5q35.1within two host genes(SLIT2and SLIT3),in a region of copy number loss,was selected as a priority candidate for follow-up as it is reported as underexpressed in lung cancer.We confirmed decreased expression of mature miR-218and its host genes by qRT-PCR in39NSCLCs relative to normal lung tissue.This downregulation of miR-218was found to be associated with a history of cigarette smoking,but not human papilloma virus.Thus,we show for the first time that putative lung cancer-associated miRNAs can be identified from genome-wide arrayCGH datasets using a bioinformatics mapping approach,and report that miR-218is a strong candidate tumour suppressing miRNA potentially involved in lung cancer.

Citation:Davidson MR,Larsen JE,Yang IA,Hayward NK,Clarke BE,et al.(2010)MicroRNA-218Is Deleted and Downregulated in Lung Squamous Cell Carcinoma.PLoS ONE5(9):e12560.doi:10.1371/journal.pone.0012560

Editor:Eric J.Bernhard,National Cancer Institute,United States of America

Received May4,2010;Accepted July21,2010;Published September3,2010

Copyright:?2010Davidson et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:This work was supported by NHMRC project grants;NHMRC Practitioner Fellowship(KF),NHMRC Research Fellowship(NH),NHMRC Career Development Award(IY),UQ Edwin Tooth PhD Scholarship(JL),Cancer Council Queensland PhD Scholarship(MD),Cancer Council Queensland Senior Research Fellowship(KF),Queensland Clinical Research Fellowship(KF,IY),The Prince Charles Hospital Foundation,Cancer Council Queensland,Smart State,and National Health and Medical Research Council.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:morgan.davidson@https://www.wendangku.net/doc/4410873645.html,.au

Introduction

MiRNAs are a highly conserved family of small non-coding RNA species that negatively regulate gene expression via RNA interference.Calin et al first identified two miRNAs(miR-15and miR-16)within13q14,a region frequently deleted in chronic lymphocytic leukaemia,with significantly reduced expression[1]. Since then,altered miRNA expression has been reported in numerous malignancies,including colon[2,3],breast[4,5],lung [6,7,8],liver[9],brain[10,11],lymphoma[12,13,14]and leukaemia[1,15,16].

In lung cancer,studies have identified the tumour suppressing let-7family and the oncogenic miR-17-92cluster of miRNAs.The let-7family is consistently downregulated in primary lung tumours and lung cancer cell lines,which abrogates let-7’s control on RAS, allowing RAS overexpression,thereby contributing to lung carcinogenesis[6,7,17].Significant overexpression of the onco-genic miR-17-92cluster of miRNAs occurs in lung cancer cell lines and tumours[18],where miR-17-92miRNAs are upregulated by oncogenic c-Myc and act as part of a regulatory network balancing cell death and proliferation with c-Myc and E2F1[19,20,21]. MiRNAs demonstrate complex patterns of genomic organisa-tion with both intergenic and intragenic miRNA genes[22,23]. Calin et al reported that most known miRNAs are in regions of genomic aberration associated with cancer[17].In keeping,let-7 and miR-17-92are linked to chromosomal deletions and gains respectively,indicating copy number variations as potential mechanisms for their dysregulation in lung tumours[17,21,24]. We reasoned that novel dysregulated lung cancer miRNAs can be identified by virtue of somatically acquired aberrant gene dosage,and this allows the large number of publically available unbiased genome-wide arrayCGH datasets to be exploited for identification of miRNAs involved in disease even though the original studies were not specifically designed for miRNA https://www.wendangku.net/doc/4410873645.html,ing this strategy we report for the first time,identification of miR-218(hsa-miR-218;MI-MAT0000275)located within a region of genomic loss(4p15.31and 5q35.1)as a putative tumour suppressor in non-small cell lung cancer (NSCLC).MiR-218was recently identified as a tumour suppressor in

cervical cancer[25]where its downregulation was linked with human papilloma virus(HPV)[25].Although we observed a significant reduction in miR-218in subjects with a history of cigarette smoking we found no relationship between HPV and miR-218in lung SCCs. Materials and Methods

Clinical Specimens

Resected primary NSCLC and corresponding normal lung tissue were obtained with informed written consent from patients undergoing lung resection at The Prince Charles Hospital between1990and2007 (Human Research Ethics Committee9124).Clinicopathological data was available for all specimens as previously published including disease recurrence[26,27]and asbestos fibre burden[28]. ArrayCGH Detection of Chromosomal Aberration ArrayCGH data for132curatively resected primary NSCLCs (training set)was provided by Dr JE Larsen(manuscript in preparation)(Table1)using the Agilent Human Genome CGH Microarray44B(Agilent,G4410B)platform and tumour sample hybridisation against normal female DNA(Human Genomic DNA Female,Promega G152A)according to the manufacturer’s instruc-tions.Filtered,normalised signal log ratios between lung tumour DNA and normal female reference DNA were used for analyses. For robust detection of DNA copy number aberrations,two independent bioinformatic approaches were used:i)Fold change (FC)of+/21.2(high discovery sensitivity)in individual probe intensity using signal log ratios(Affymetrix,Statistical Algorithms Reference Guide);and ii)CGH Explorer Analysis of Copy Errors (ACE)algorithm[29]controlling significance and false discovery rate at,0.001to avoid Type1error for multiple comparisons. Adenocarcinomas(AC)(n=72)and squamous cell carcinomas (SCC)(n=60)were analysed separately.Analysis of chromosome X was conducted in the female data set(AC n=23and SCCs n=17).Chromosome Y,which does not contain any known miRNA genes,was not examined.Selected thresholds for all analyses are summarised in Table S1.

Identifying miRNAs within Regions of ArrayCGH Detected Chromosomal Aberration

474human miRNAs from Sanger miRBase Version9.0 (https://www.wendangku.net/doc/4410873645.html,/)[30,31,32]were physically mapped onto the arrayCGH dataset using miRNA precursor chromosome number and nucleotide position,effectively position-ing them between proximal and distal flanking arrayCGH probes. MiRNA genes were classified as having copy number aberrations if both flanking arrayCGH probes were:i)located within35kb(average spatial resolution of the platform)of the miRNA or positioned within the miRNAs host gene;and ii) identified as concordant gain or loss by both bioinformatic methods(i.e.FC and ACE).These were then called as ‘‘significant’’if both flanking probes were concordantly altered in$25%but discordant in,10%of primary tumours.This produced a list of miRNAs in regions of copy number variation. Validation of miRNAs from Independent Test Sets

We also interrogated published arrayCGH data from three independent primary NSCLC cohorts[33,34,35](Table S2). Regions of chromosomal aberration were compiled and miRNAs in areas of gain or loss were identified using genomic positioning information as described above.This list of miRNAs was then directly compared with candidates derived from the TPCH (training)dataset to select those miRNAs within regions of aberration consistent in the training set and at least one test set. Prioritising miRNAs with Gene Dosage and Expression Concordance

To select candidate miRNAs with concordantly altered dosage and expression,we interrogated four public datasets of mature miRNA expression from independent NSCLC cohorts[6,7,8,36] (Table S3).Candidate miRNAs with changes in expression that were concordant with their inferred copy number changes were prioritised for further study.

Biological Verification of Aberrant miRNA Expression Mature miRNA expression was measured using TaqMan qRT-PCR assays(Applied Biosystems)in39paired NSCLCs and normal lung(SCCs n=18and ACs n=21)(Table2).Total RNA was isolated from30mg of tissue using TRIzol(Invitrogen)and DNase treated(Ambion).MiRNA-specific reverse transcription for miR-218and U6snRNA(internal control)used stem-loop TaqMan primer/probe sets for real-time PCR(qRT-PCR)[37]. Triplicate TaqMan qRT-PCR assays were performed on a Corbett Research Rotor-Gene6000with reference total RNA

Table1.Clinicopathological characteristics of arrayCGH cohort.

Squamous Cell Carcinoma(n=60)Adenocarcinoma(n=72)All Primary NSCLCs(n=132)

Age(years)66.2

(38.9–83.7)64.7

(35.9–81.6)

65.4

(35.9–83.7)

Sex Male434992

Female172340

Stage Stage I295382

Stage II231033

Stage III7512

Stage IV145

Smoking Status a Current263056

Former313667

Never369

a Former smokers had quit smoking at least one year prior to lung resection,while current smokers continued smoking within one year of lung resection.Pack years were calculated as number of cigarettes per day,multiplied by number of years of smoking,divided by20.Never smokers had smoked less than100cigarettes in their life-time. doi:10.1371/journal.pone.0012560.t001

certified to contain miRNAs(FirstChoice Human RNA Survey Panel,Ambion).Reaction efficiency and linear dynamic range was determined for each assay.A ratio of miRNA expression to U6 snRNA expression was obtained using the Pfaffl method[38]. Correlating Dysregulated miRNA and Host Gene Expression

Host gene expression was measured by qRT-PCR using SYBR Green chemistry in the same cohort of39NSCLCs.DNase treated total RNA were reverse transcribed using Superscript III (Invitrogen).Primers were designed using Primer Express(Applied Biosystems)and are listed in Table S4.Standard curves were performed to determine the linear dynamic range and reaction efficiency.All reactions were performed in triplicate on a Corbett Rotorgene6000with melt curve analysis to confirm detection of a single amplicon,with Universal Human Reference RNA(Strata-gene)as reference.Relative gene expression was calculated and normalised against internal controls(18S rRNA,ACTN4and BAT1)[39],using the Pfaffl method[38].

HPV Testing

Presence of HPV DNA was assessed in a cohort of72lung SCCs accessed from TPCH Tumour Bank,including the18cases with miR-218expression data.HPV testing was performed by Gribbles Pathology,Victoria,Australia,using the Genera Biosystems PapType RUO Kit which detects14high-risk(16, 18,31,33,35,39,45,51,52,56,58,59,66and68)and two low risk(6and11)HPV subtypes.This assay is based on2rounds of PCR amplification of HPV and control gene DNA(alkali myosin light chain protein)with addition of a fluorescent reporter dye and utilises silica detection beads which fluoresce when hybridised to its specific target DNA.

Statistical Analysis

Statistical analyses were performed as above or with SPSS Version13.0(SPSS Inc Chicago,IL,USA)using Mann-Whitney U and Pearson correlations.p values,0.05were considered statistically significant.Survival analysis was undertaken with log rank statistic of constructed Kaplan-Meier curves.

MiRNA Target Prediction and Pathway Analysis Predicted mRNA targets of miR-218were identified by using four online miRNA target prediction programs:PicTar(5species conservation)[40],TargetScan4.1[41,42,43],miRBase Targets Version5[30,31,32]and miRNAMap 2.0[44,45].Over-represented gene ontologies(GO)and functional classes for putative target genes of miR-218predicted by two or more of the algorithms were identified using the Database for Annotation,

Table2.Clinicopathological characteristics of miR-218validation cohort.

Squamous Cell Carcinoma Adenocarcinoma All Primary NSCLCs

(n=18)(n=21)(n=39)

Age(years)66.567.667.1

(range)(44.4–83.7)(47.3–81.6)(44.4–83.7)

Sex Male121426

Female6713

Stage Stage I51520

Stage II11213

Stage III235

Stage IV011

Smoking Current7714

Status a Former81119

Never336

Asbestos Exposure Exposed(.20AB/gww)51015

Low Exposure(1–20AB/gww)011

No Exposure(0AB/gww)131023

HPV DNA HPV160––

HPV181––

HPV311––

Negative16––

Recurrence Rec(3–18months)71219

Non-Rec(.36months)8715

Excluded325

Survival Alive7613

Deceased111526

Survival Duration(months)(range)24.7(6.6–62.4)25.3(4.3–84.6)25.1(4.3–84.6)

a Former smokers had quit smoking at least one year prior to lung resection,while current smokers continued smoking within one year of lung resection.Pack years were calculated as number of cigarettes per day,multiplied by number of years of smoking,divided by20.Never smokers had smoked less than100cigarettes in their life-time.Abbreviations:Rec,Recurrence;Non-Rec,Non-Recurrent.

doi:10.1371/journal.pone.0012560.t002

Visualization and Integrated Discovery(DAVID,April2008 release)gene-GO enrichment analysis(ranked by the Expression Analysis Systematic Explorer(EASE)score threshold)and gene functional classification(performed with the highest stringency setting)[46,47].Ingenuity Pathway Analysis(Ingenuity H Systems, https://www.wendangku.net/doc/4410873645.html,)was performed on both the entire miR-218 target gene list and a prioritised list of miR-218targets found to be enriched$2.0by DAVID gene functional classification. Results

MiRNAs are Located in Regions of Genomic Alteration in Lung Cancer

From TPCH arrayCGH data(training set),89of474(19%) miRNAs were within chromosomal regions deleted(41miRNAs) or gained(48miRNAs)in132primary NSCLC(Table S5). Within the SCC subtype,77miRNAs were associated with DNA copy number changes,with40miRNAs in areas of gain and37in regions of loss.Twenty-six miRNAs were identified for ACs,with 21and five miRNAs in regions of gain and loss respectively. Fourteen miRNAs were located within regions of copy number variation common to both SCC and ACs(one associated with DNA loss and13associated with DNA gain).On the X chromosome,five and four miRNAs were found to be associated with regions of loss in ACs and SCCs respectively.

Validation of Candidate NSCLC miRNAs from Public Data Test Sets

MiRNA dosage.To validate candidate miRNAs,we repeated the positional mapping analysis using published independent arrayCGH data test sets[33,34,35]and found51miRNAs;28 and23miRNAs located within regions of gain or loss respectively (Table S6).Eighteen of these were among the89miRNAs identified from positional mapping to arrayCGH(Figure1).No miRNAs were found to be located in genomic regions of copy number variation

that were discordant between our cohort(training set)and published arrayCGH cohorts(test sets).

MiRNA Expression.To enrich for miRNAs with concordant dysregulated expression and dosage,we reviewed the literature and found80mature miRNAs reported to have altered expression levels(44increased,32decreased and four with conflicting reports) in primary NSCLCs[6,7,8,36],16were represented in the89 miRNA set with10showing expression dysregulation concordant with predicted miRNA gene copy number change.Seven miRNAs were among the51miRNAs identified from published arrayCGH studies,but only three had concordant miRNA expression and dosage between the public datasets.Two miRNAs,miR-218and miR-216,were common to training and all test sets,however,only miR-218demonstrated concordant loss of copy number and expression(Figure1).Expression levels of38/89candidate NSCLC miRNAs and19/51published arrayCGH NSCLC miRNAs have only recently been annotated in miRBase and have not yet been studied in lung cancer.

MiR-218Host Genes are in Regions of Copy Number Loss in NSCLC

MiR-218is produced from two unique miRNA precursors(hsa-mir-218-1;MI0000294and hsa-mir-218-2;MI0000295),encoded in separate genomic locations,4p15.31and5q35.1respectively. Both precursors are intragenic,with mir-218-1situated within intron15–16or14–15of SLIT2(Slit Homolog2)and mir-218-2 residing within intron14–15of SLIT3(Slit Homolog3)(Figure1). Mir-218-2was identified as within a region of copy number loss in .25%of SCCs but only.15%of ACs with average fold losses of SLIT3flanking probes21.205(range22.23to21.11)and21.115 (range21.85to+1.39)respectively.However,mir-218-1was located in a region of loss unique to SCCs,with an average fold loss of SLIT2flanking probes21.13(range21.95to+1.29)(Table S7). MiR-218Expression is Reduced in NSCLCs Compared with Paired Normal Lung

To confirm decreased miR-218dosage and expression in lung cancer,we measured mature miR-218expression in21ACs and 18SCCs from our arrayCGH training set and their paired normal lung.MiR-218expression was down-regulated in85%(33/39)of NSCLC tumours compared with paired normal lung.Statistically significant decreases were observed in both SCCs(mean FC=24.4,p,1.0e-4)and to a lesser extent ACs(mean FC=22.0,p=0.001)(Figure2a).

Reduced miR-218Expression is Associated With SLIT2 and SLIT3Copy Number Loss

Correlation of miR-218expression with host gene(SLIT2and SLIT3)arrayCGH probe copy number found the majority of samples(31/39,79.5%)demonstrated miR-218down-regulation with a decrease in SLIT2and/or SLIT3copy number however the correlation was not significant(p.0.05;Pearson)(Figure S1a). Complete concordance,that is,a reported loss of both host genes plus a decrease in miR-218expression,was observed in55.6% (10/18)SCCs and38.1%(8/21)of

ACs.

Figure 1.Identification of miR-218from concordant copy number variation and gene expression from independent data sets.Expression levels of38/89candidate miRNAs and19/51published arrayCGH miRNAs have not been studied due to their recent inclusion in miRBase.The genomic location of miR-218within its two host genes, SLIT2and SLIT3,is also illustrated.

doi:10.1371/journal.pone.0012560.g001

Concordant Downregulation of miR-218,SLIT2and SLIT3mRNA Expression

To confirm predicted host gene down-regulation,we measured SLIT2and SLIT3expression in 37of the 39NSCLC samples and compared it with their paired normal lung tissue (19ACs and 18SCCs).As expected,SLIT2and SLIT3down-regulation was observed in 36/37and 34/37NSCLCs respectively,with statistically significant down-regulation of both SLIT2and SLIT3expression in both ACs and SCCs (p ,0.001)(Figure 2b).

SLIT2copy number loss plus reduced expression was observed in 83%(15/18)SCCs and 79%(15/19)AC.Whilst SLIT3demonstrated copy number and expression concordance in 83%(15/18)SCCs but only 53%(10/19)ACs.For both SLIT2and SLIT3,the relationship between host gene expression and copy number was not significant (p .0.05;Pearson)(Figure S1b).

To determine whether this intragenic miRNA is co-regulated with host gene expression,we examined miR-218and SLIT2/SLIT3expression levels.Concordant down-regulation of miR-218,SLIT2and SLIT3was observed in 15/18(83.3%)SCCs and 15/19(79%)ACs.Notably,one AC had concordant increases in miR-218,SLIT2and SLIT3expression with CN loss of only SLIT3.In SCCs,miR-218was moderately correlated with SLIT2(R =0.496,p =0.036)and SLIT3(R =0.459,p =0.055),however,only SLIT2was statistically significant.For ACs,weak-moderate correlations were detected (SLIT2:R =0.378,p =0.111;SLIT3:R =0.297,p =0.216)but these were not statistically significant (Figure S1c).

MiR-218,SLIT2and SLIT3Downregulation is Associated with SLIT2and SLIT3Gene Dosage

Next,we compared SLIT2and SLIT3copy number with miR-218,SLIT2and SLIT3expression to determine if their expression was due to gene dosage.The 15ACs and 15SCCs found to have concomitant reduced miR-218and host gene expression also demonstrated copy number loss in SLIT2and/or https://www.wendangku.net/doc/4410873645.html,plete concordance (loss of both host genes plus a decrease in miR-218expression)between SLIT2and SLIT3copy number and miR-218,SLIT2and SLIT3expression,was observed in 9/19(47.4%)ACs and 10/18(55.6%)SCCs (Figure S2and Table S8).

For one AC and two SCCs,miR-218expression was increased despite reduced host gene copy numbers and expression.

Association of miR-218with Clinicopathological Phenotypes

Smoking History.

Compared to normal lung tissue,miR-218expression was significantly decreased in tumours of current (SCC:FC-4.4;AC:FC-4.1;p ,0.05)and former (SCC:FC-5.1;AC:FC-1.9;p ,0.05)smokers for both subtypes of NSCLC (Figure 3a)but no decrease was observed in never smokers (albeit small sample:SCC n =3,ACs n =3).No significant associations were demonstrated between miR-218expression and total number of pack years smoked (in current and former smokers)or time since quitting smoking (former smokers)in SCCs or ACs (data not shown).

HPV Status.A large cohort of SCCs (n =72)were screened to detect the presence of HPV DNA,with 9%(8/72)of these lung SCCs testing positive.HPV subtypes identified were potentially oncogenic,including types 16(4/8),18(3/8)and 31(1/8).For 18SCCs with miR-218expression data,9%(2/18)tested positive for HPV (subtypes 18and 31)(Table 2)and HPV status not found to be associated with a reduction in miR-218expression (Figure 3b).Asbestos.Altered miR-218expression in NSCLC was also examined in relation to asbestos exposure.Asbestos fibre burden has previously been assessed for this cohort,and tumours with .20asbestos bodies per gram wet weight lung tissue (AB/gww)were considered ‘asbestos exposed’and those with 0AB/gww were considered to be ‘not exposed’[28].Altered miR-218expression was not found to be associated with asbestos exposure (Figure 3c)nor asbestos fibre burden (number of AB/gww lung tissue -data not shown).

Recurrence and Survival.Following the earlier publication by Larsen et al [26,27],recurrent NSCLCs were those that had recurred within 3–18months post-surgical resection and non-recurrent subjects were those who had remained disease free for a period of at least 36months post-resection.No association was observed between miR-218expression levels and disease recurrence or NSCLC survival post-resection (Figure

3d–e).

Figure 2.Expression of miR-218and its host genes in primary NSCLCs and their paired normal lung.*p ,0.05;**p ,0.01.(a)Down-regulation of miR-218expression was observed in both ACs (FC -2;p =0.001;Z score =23.258)and SCCs (FC 24.365;p ,0.0001;Z score =24.406).(b)A reduction in host gene expression was observed in both ACs (SLIT2:FC 212.77,p ,0.0001,Z score =24.277;SLIT3:FC 213.73,p ,0.0001,Z score =23.839)and SCCs (SLIT2:FC 210.85,p ,0.0001,Z score =25.182;SLIT3:FC 25.46,p ,0.0001,Z score =24.540).Abbreviations:FC,Fold change.

doi:10.1371/journal.pone.0012560.g002

Putative Targets of miR-218Support its Role in Carcinogenesis

We identified predicted target genes of miR-218using four online miRNA target prediction algorithms.PicTar [40]and miRNAMap 2.0[44,45]identified 575and 688target genes respectively,with 645conserved sites and 133poorly conserved sites.TargetScan 4.1[41,42,43]and miRBase Targets V5[30,31,32]reported 570and 946miR-218target sites respectively,where multiple target sites could be present in the one gene.In total,1794individual target genes were identified by these four target prediction algorithms (578identified by two or more of the algorithms)(available on request).

Gene ontologies and biological function of the 578target genes were explored using DAVID (April 2008Release)to determine biological relevance [46,47].GO enrichment analysis revealed that biological processes such as cell adhesion,protein modifica-tions and transport,development and cell signalling were over-represented amongst miR-218targets (p ,0.001).Over-represent-ed molecular functions (p ,0.01)were predominantly focused on protein binding function,in particular to actin,cytoskeletal proteins,cyclic nucleotides,protein phosphatase 2A,cAMP,and metal ions.Similar to gene ontology analysis,functional annotation clustering reflected enrichment of groups of genes largely involved in cell adhesion (enrichment score =4.66),protein modification relating to ubiquitin cycle (enrichment scores =3.67and 3.33)or kinase activity (enrichment score =2.88),and regulation of transcription (including proto-oncogenes,enrichment score =2.22)(Table S9).

Network analysis of the 578target genes by Ingenuity Pathway Analysis identified 39gene networks involved in biological functions such as cell-to-cell signalling,amino acid metabolism,post-translational modifications,cancer and respiratory system development and function (p ,0.05)(Figure S3a and Table S10).Canonical pathway analysis found numerous cancer signalling pathways including Wnt/b -catenin signalling,ERK/MAPK signalling and Notch signalling (p ,0.05)(Figure S3b).To attempt to define a more specific network or pathway associated with miR-218,we performed a focused analysis using 121enriched genes ($2.00)identified with functional annotation clustering in DAVID.This identified 10gene networks that reflected similar cancer-related biological functions to those identified using the larger target gene list (Table S10).For instance,in the ‘gene expression,cancer and cell morphology’network,miR-218may target genes directly and indirectly linked to two oncogenes,MYC and SRC (Figure S4).

Discussion

We demonstrate that genomic profiling can be used to identify cancer related miRNAs.We found 19%of miRNAs were located in regions of copy number variation in primary NSCLC,which is relatively lower than ovarian (37%),breast (73%)and melanoma (86%)[48].Aside from being different tumour types,this may be due to more recently identified miRNAs not highly represented in cancer-associated genomic regions,our high stringency bioinfor-matics,or both.We limited ourselves to miRNAs located

within

Figure 3.Relationship of miR-218expression to smoking (a),HPV (b),asbestos (c),recurrence (d)and survival (e).*p =0.05;**p =0.01.Abbreviations:FC,Fold change.Statistically significant downregulation of miR-218expression was observed in current and former smokers of both NSCLC subtypes.No significant associations were observed between miR-218expression and HPV,asbestos,disease recurrence or survival.

doi:10.1371/journal.pone.0012560.g003

host genes or next to flanking probes that were within the spatial resolution of the platform(35kb),because the array used in this study did not have any probes directly representing any miRNA genes.Next generation higher-resolution arrayCGH platforms will refine this approach specifically detecting miRNA genomic loci. Nonetheless,we successfully identified89potential lung cancer miRNAs,including established oncogenic and tumour suppressing miRNAs.Three members of the known tumour suppressing let-7 family of miRNAs were identified in regions of loss(let-7g,let-7f-2 and miR-98),and oncogenic miR-21was found within a genomic region of amplification.

Over half of the89miRNAs(65%)are intragenic;interestingly, many of the host genes have reported roles in cancer.For example, MCM7(minichromosome maintenance protein7)is the host gene for three of the miRNAs(miR-25,miR-93and miR-106b)we identified and has been reported to be amplified or over-expressed in human malignancies including prostatic,pancreatic,thyroid, cervical and colorectal cancers[49,50,51,52,53,54].In prostate cancer,increased levels of these3miRNAs occurred with amplified and over-expressed MCM7[55].This observation supports the idea proposed that miRNAs and their host genes may be jointly affected by copy number changes[48].

Only one of the89miRNAs,miR-218,demonstrated concordant changes in copy number and expression.Additional candidates may be identified with publication of new miRNA expression studies in NSCLC as half of the miRNAs with concordant copy number changes(10/18)were excluded from this first pass analysis as their mature expression levels have not been measured.Furthermore, miRNAs are subject to complex regulatory mechanisms so it is not surprising to observe numerous miRNAs with discordant copy number and expression(such as miR-216).However,as with any genome-wide approach for gene discovery,there is the potential to identify false positives.We have attempted to alleviate this risk by using high stringency criteria for identification of candidate miRNAs from our aCGH data and subsequently using published NSCLC cohorts with copy number and miRNA expression data to independently validate these candidates.As such,miR-218emerged as a strong candidate tumour suppressor,within a region of copy number loss in greater than two NSCLC studies and with demonstrated loss of expression in a third independent cohort. Produced from two separate precursors,miR-218is found within host genes SLIT2and SLIT3,with both the miRNA and its host genes demonstrating reduced expression in the majority (80%)of NSCLCs.Zhang et al reported copy number losses of mir-218-1and SLIT2in ovarian(16%),breast(36%)and melanoma (33%),however,the alternative precursor and host gene were not mentioned[48].It has been suggested that intronic miRNAs share host gene transcriptional regulatory control with resulting co-expression[22,23].Despite noting a reduction in both miR-218 and host gene expression in the majority of NSCLCs,only a moderate correlation between miR-218and SLIT2expression was detected,with no significant relationship with SLIT3.This may be a reflection of disproportionate production of miR-218from each genomic site,alterations in miRNA biogenesis or miR-218may have a promoter separate from its host gene.Similarly,no significant associations between miR-218or host gene expression with host gene copy number were identified,but we found that complete concordance(copy number loss of both host genes with reduced miRNA and host gene expression)observed in51%of lung cancers studied.These findings would suggests that genetic loss may contribute to the observed reduction in miR-218,SLIT2 and SLIT3expression,however it is likely that aberrations in multiple regulatory mechanisms are involved,including alterations to miRNA biogenesis and epigenetic controls.For example,altered epigenetic control of SLIT2and SLIT3has also been reported in lung cancer with hypermethylation of SLIT2and to a lesser extent SLIT3[56,57].Thus,epigenetic silencing is an alternative or additional‘hit’,and strengthens the case for miR-218and its host genes as potential tumour suppressor genes. Additional support for a role for miR-218in NSCLC comes from the potential association between miR-218downregulation and cigarette smoke exposure.Further,Schembri et al has also demonstrated a link between miR-218downregulation and smoking;exposing human bronchial epithelial cells to cigarette smoke extract decreased miR-218expression levels[58].Our observation that in both subtypes of NSCLC,miR-218expression is significantly reduced in subjects with a history of cigarette smoking,provides additional support the notion that miR-218 may be involved in tobacco-related carcinogenesis[58].However, the number of never smokers used in this study is insufficient to confirm this relationship and further investigation is required with larger cohorts and functional validation to explore a causative relationship.We also examined whether the decrease in miR-218 expression was associated with asbestos,a well known lung carcinogen,but found no significant correlation.As miR-218may play a role in metastasis,with Leite et al reporting high levels of miR-218in high grade prostate cancer compared to significantly reduced levels in metastatic prostate cancers[59],we examined the survival of our cases with miR-218dysregulation.In lung cancer,we found no relationship between miR-218expression levels and NSCLC recurrence or patient survival.

MiR-218has also been shown to be downregulated in cervical carcinoma through the action of the HPV16E6oncogene and may be important in early cervical tumourigenesis[25].We have previously identified a limited association for HPV infection in lung cancer,with reports of HPV prevalence ranging from5–22%[60,61]. We did not find any link between HPV status and miR-218expression but one reason may be that neither of our HPV positive samples were HPV type16,and reduced miR-218has only been reported with the HPV16–E6oncogene.However,the small numbers preclude strong conclusions and more samples need testing.MiR-218has also been reported to be reduced in gastric cancer,with reduced expression linked to Helicobacter pylori infection and carcinogenesis[62]. Target prediction found miR-218can target numerous oncogenes,such as KIT,RET,BCL9,DCUN1D1and PDGFRA. Gene ontology and functional annotation clustering of the predicted targets revealed enrichment of genes involved in cell adhesion,protein modification,development,cell signalling and regulation of transcription,all processes that may contribute to carcinogenesis.These findings were reflected in the Ingenuity Pathway Analysis,with miR-218potentially regulating genes involved in cancer-related biological functions and three recog-nised cancer signalling pathways.Focused pathway analysis also revealed that miR-218may regulate genes directly and indirectly related to MYC and SRC,two well characterised oncogenes [20,21,63,64,65].Recent studies have begun to authenticate miR-218target genes,including LAMB3(laminin-5b3)[25],ECOP (Epidermal growth factor receptor-coamplified and overexpressed protein)[58,62]and transcription factor MAGF(v-maf musculo-aponeurotic fibrosarcoma oncogene homolog G)[58].

In summary,arrayCGH cancer datasets are increasingly available with unbiased probes or selected cancer associated genes.We combined bioinformatic and experimental approaches to test whether miRNAs located in regions of gene dosage alteration had concordant altered expression,and therefore of importance.With this approach we identified putative tumour suppressor miR-218, experimentally confirmed its downregulation in NSCLC,and provided additional support for an association between reduced

expression and cigarette smoke exposure.In addition,we identified relevant cancer-related target genes and pathways targeted by miR-218,supporting a potential role as a tumour suppressor gene for NSCLC,especially SCCs.Nonetheless,further evidence including structural mutations and demonstrable tumour suppressor functions should be gathered before miR-218can be appropriately designated as a tumour-suppressor gene for lung cancer.

Supporting Information

Figure S1Relationship between host gene copy number,host gene expression and miR-218expression in lung adenocarcinomas and squamous cell carcinomas.*p=0.05.(a)No significant Pearson correlation was observed for host gene copy number and miR-218expression in SCCs(SLIT2:20.282,p=0.257; SLIT3:20.111p=0.661)or ACs(SLIT2:20.078p=0.737; SLIT3:20.107,p=0.646).(b)No significant Pearson correlation was observed for host gene copy number and expression in SCCs (SLIT2:20.389,p=0.111;SLIT3:20.265p=0.288)or ACs (SLIT2:0.009p=0.970;SLIT3:0.249,p=0.304).(c)A significant Pearson correlation was observed between miR-218 expression and SLIT2expression in SCCs(0.496,p=0.036)but not ACs(0.378,p=0.111).No significant correlation was observed between miR-218expression and SLIT3expression(SCC:0.459, p=0.055;AC:0.297,p=0.216).

Found at:doi:10.1371/journal.pone.0012560.s001(1.42MB TIF) Figure S2MiR-218,SLIT2and SLIT3expression and SLIT2 and SLIT3copy number in lung squamous cell carcinomas(a)and adenocarcinomas(b).Complete concordance(loss of both host genes plus a decrease in miR-218expression)between SLIT2and SLIT3copy number and miR-218,SLIT2and SLIT3expression, was observed in9/19(47.4%)ACs and10/18(55.6%)SCCs.For one AC and two SCCs,miR-218expression was increased despite reduced host gene copy numbers and expression.

Found at:doi:10.1371/journal.pone.0012560.s002(0.30MB TIF) Figure S3Ingenuity Pathway Analysis miR-218.Core analysis for578predicted miR-218target genes.(a)Top20biological functions.Threshold bar is set at significance of p,0.05and-log(p-value)of1.3.(b)Top20canonical pathways.Threshold bar is set at significance of p,0.05and-log(p-value)of1.3.Line illustrates the ratio of miR-218target genes in the pathway divided by the total number of genes in the pathway.Red circles highlight cancer-associated functions or pathways.

Found at:doi:10.1371/journal.pone.0012560.s003(2.24MB TIF) Figure S4Network of enriched miR-218target genes linked to the biological functions of gene expression,cancer and cell morphology identified from Ingenuity Pathway Analysis.MiR-218 may target genes(coloured orange)directly and indirectly linked to two well known oncogenes,MYC and SRC(coloured red).For clarity,additional miRNAs and their targets involved in this network were removed therefore only9of the11miR-218targets are shown.RELN is also a predicted target of miR-218but was not included in the enriched gene list.

Found at:doi:10.1371/journal.pone.0012560.s004(2.29MB TIF) Table S1Selected thresholds for identification of chromosomal aberrations in arrayCGH data.Abbreviations:SCC,Squamous Cell Carcinoma;AC,Adenocarcinoma;FC,Fold change;ACE, Analysis of Copy Errors;FDR,False Discovery Rate.

Found at:doi:10.1371/journal.pone.0012560.s005(0.03MB DOC)

Table S2ArrayCGH Publications used for miRNA prioritisa-tion.Abbreviations:SCC,Squamous Cell Carcinoma;AC,Adenocarcinoma;AdSq,Adenosquamous Carcinoma;BAC, Bronchioalveolar Carcinoma;SCLC,Small Cell Lung Carcino-ma;LC,Large Cell Carcinoma.

Found at:doi:10.1371/journal.pone.0012560.s006(0.03MB DOC)

Table S3MiRNA Expression studies used for miRNA prior-itisation.Abbreviations:SCC,Squamous Cell Carcinoma;AC, Adenocarcinoma;AdSq,Adenosquamous Carcinoma;LC,Large Cell Carcinoma.

Found at:doi:10.1371/journal.pone.0012560.s007(0.04MB DOC)

Table S4Primers for host gene qRT-PCR

Found at:doi:10.1371/journal.pone.0012560.s008(0.03MB DOC)

Table S5Candidate miRNAs identified from positional ar-rayCGH analysis.Abbreviations:AC,Adenocarcinoma;SCC, Squamous Cell Carcinoma.

Found at:doi:10.1371/journal.pone.0012560.s009(0.12MB DOC)

Table S6Candidate miRNAs identified from public arrayCGH data.

Found at:doi:10.1371/journal.pone.0012560.s010(0.07MB DOC)

Table S7Summary of arrayCGH analysis for mir-218-1and mir-218-2.

Found at:doi:10.1371/journal.pone.0012560.s011(0.07MB DOC)

Table S8Summary of miR-218expression and SLIT2/SLIT3 expression and copy number changes.Abbreviations:FC,Fold Change;SCC,Squamous Cell Carcinoma;AC,Adenocarcinoma. Found at:doi:10.1371/journal.pone.0012560.s012(0.07MB DOC)

Table S9Enriched miR-218target gene groups identified by gene functional classification in DAVID.

Found at:doi:10.1371/journal.pone.0012560.s013(0.03MB DOC)

Table S10Top10networks for predicted miR-218target genes from ingenuity pathway analysis.

Found at:doi:10.1371/journal.pone.0012560.s014(0.03MB DOC)

Acknowledgments

Sincere thanks to the patients of The Prince Charles Hospital for their participation in this study,and The Prince Charles Hospital surgeons, pathologists,scientists and Thoracic Research Laboratory staff for their assistance with specimen collection.

Author Contributions

Conceived and designed the experiments:MRD JEL IAY NKH RVB KMF.Performed the experiments:MRD JEL.Analyzed the data:MRD IAY NKH RVB KMF.Contributed reagents/materials/analysis tools: JEL.Wrote the paper:MRD.Study design,bioinformatic and data analyses,technical validation,target predication analyses and manuscript preparation:MRD.Performed arrayCGH experiments and data pre-processing,project plan and bioinformatics advice:JEL.Study design and assistance with bioinformatics and statistical analyses:IAY NKH.Sample collection and pathological staging:BEC EED.Patient recruitment and consent,collection of clinicopathological data:LHP.Study design, assistance with development of methods for bioinformatic analyses:RVB. Study design,data analysis and manuscript revision:KMF.

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英语各种词性的用法

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的“稀有情愫”——敬爱。 有时候，为了形容家的美好，我们试图用一些华丽的、带着刺眼光芒的词语去修饰它，但这样，其实只是走远了它。只要一家人在一起，这就是家的样子，朴实且真切。林爷爷笔下的家，就是平凡的。三个小孩就是三个太阳。樱樱、琪琪和玮玮，虽然经常调皮，不免惹是生非，可一次平息的怒火，就是一片的温暖光芒。供稿：六（3）班郑雯欣 感谢阅读，希望能帮助您！

英文各种词性的用法知识

精品文档 英文各种词性的用法及句子结构讲解： 十大词类： 1, 名词，Nou ns (n.) 表示人或事物的名称 box, pen, tree,apple 2, 代词，Pronouns (pron.) 代替名词、数词、形容词 We, this, them,myself 3，形容词，Adjectives(adj.) 用来修饰名词，表示人或事物的特征good, sad, high, short 4，数词，Numerals(num.)表示数目或顺序 one,two, first 5，动词，Verb (v.) 表示动作或状态Jump,sing,visit vt.是及物动词，vt.后必须跟宾语：sing a song vi.是不及物动词，vi.后不直接带宾语或不带宾语:jump high 6，副词，Adverbs (adv.) 修饰动、形、副等词，表示动作特征there,widely,suddenly 7，冠词，Articles (art.) 用在名词前，帮助说明名词所指的范围a, an, the 8，介词，Prepositions (prep.) 用在名词或代词前，说明它与别的词的关系in,on,down,up 9，连词，Conjunctions (conj.) 表示人或事物的名称 if,because,but 10，感叹词，In terjectio ns (in t.) 代替名词、数词、形容词等 oh,hello,hi,yeah 组成句子的各个部分叫句子成分。英语句子成分有主语，谓语，表语，宾语，宾语补足语，定语，状语等。 顺序是主语，谓语，宾语，宾语补足语，而表语，定语，状语的位置要根据情况而定。 1、主语 主语表示句子主要说明的人或事物，一般由名词，代词，数词，不定式等充当。 2、谓语 谓语说明主语的动作，状态或特征。 一般可分为两类： 1) ，简单谓语 由动词(或短语动词)构成。 可以有不同的时态，语态和语气。 2) ，复合谓语：情态动词+动词原形 3、表语 表语是谓语的一部分，它位于系动词如be之后，说明主语身份，特征，属性或状态。一般由名词，代词，形容词，副词，不定式，介词短语等充当。 4、宾语 宾语表示动作行为的对象，跟在及物动词之后，能作宾语的有名词，代词，数词，动词不定式等。 有些及物动词可以带两个宾语，往往一个指人，一个指物，指人的叫间接宾语，指物的叫直接宾语。有些及物动词的宾语后面还需要有一个补足语，意思才完整，宾语和它的补足语构成复合宾语。 5、定语 在句中修饰名词或代词的成分叫定语。 用作定语的主要是形容词，代词，数词，名词，副词，动词不定式，介词短语等。形容词，代词， 数词，名词等作定语时，通常放在被修饰的词前面。 但副词，动词不定式，介词短语等作定语时，则放在被修饰的词之后。

简单句的翻译训练

基本句型汉译英练习主系表结构 本结构是由主语+系动词+表语组成，主要用以说明主语的特征，类属，状态，身份等。 系动词有 （1）表示特征和存在状态的 be, seem, feel, appear, look, smell, taste, sound. （2）表示状态延续的 remain, stay, keep, continue, stand. （3）表示状态变化的 become, get, turn, go, run, fall, come, grow. 翻译练习： 1 我的兄弟都是大学生。 2 冬季白天短，夜晚长。 3 布朗夫人看起来很健康。 4 十五岁他就成为有名的钢琴家了。 5 孩子们，请保持安静。 6 这本书是有关美国历史的书。 7 她的工作是在幼儿园里照看儿童。 8 他失业了。 9 树叶已经变黄了。 10 这个报告听起来很有意思。 基本句型汉译英练习主谓宾结构（一） 此结构是由主语+谓语+宾语够成。其中的谓语动词须是及物的动词或及物的动词词组。 宾语须是名词或相当于名词的成分。 1 昨晚我写了一封信。 2 今天下午我想同你谈谈。 . 3 这本书他读过多次了。 4 他们成功地完成了计划。 5 你们必须在两周内看完这些书。 6 那位先生能流利地说三种语言 7 我收到了笔友从澳大利亚寄来的信。 8 Jim 还不会自己穿衣服。 9 我们大家都相信Jack 是一个诚实男孩。 10 他不知道说什麽好。 11 他每天早晨洗冷水澡。 12 我开窗户你在意吗？ 主谓宾结构（二） 此结构中的谓语动词常常是动词词组,分两种情况: 1.及物动词+副词， 2.不及物动词+介词。 1.含有away, out, forward, up 等副词的动词词组是可拆分的。如， They carried out the plan successfully.我们还可以说， They carried the plan out successfully. 但我们只能说 As the plan was practical, they carried it out successfully. 动副词组都可以这样用，如，point out（指出）, carry out（执行）, put forward（提出）, work out （做出，算出）, find out（找出）, give up（放弃）, give away（赠送，分发）,pick up（拣起）, put up