Aberrant Expression Profile of Long Noncoding RNA in Human ...

Research ArticleAberrant Expression Profile of Long Noncoding RNA in HumanSinonasal Squamous Cell Carcinoma by Microarray Analysis

Ling-zhao Meng1 Ju-gao Fang2 Jing-wu Sun1 Fan Yang1 and Yong-xiang Wei1

1Department of Otolaryngology-Head and Neck Surgery Beijing Anzhen Hospital Capital Medical University Beijing 100029 China2Department of Otolaryngology-Head and Neck Surgery Beijing Tongren Hospital Capital Medical University Beijing 100730 China

Correspondence should be addressed to Ju-gao Fang fangjugaotongren163com andYong-xiang Wei weiyongxianganzhen163com

Received 12 September 2016 Accepted 8 November 2016

Academic Editor Jan Plzak

Copyright copy 2016 Ling-zhao Meng et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

ObjectivesThis study aimed to identify aberrantly expressed long noncoding RNAs (lncRNAs) profile of sinonasal squamous cellcarcinoma (SSCC) and explore their potential functions Methods We investigated lncRNA and mRNA expression in SSCC andpaired adjacent noncancerous tissues obtained from6 patients withmicroarrays Gene ontology (GO) analysis and pathway analysiswere utilized to investigate the gene function Gene signal-network and lncRNA-mRNA network were depicted Quantitativereal-time polymerase chain reaction (qRT-PCR) was utilized to validate 5 lncRNAs in a second set of paired SSCC and adjacentnoncancerous tissues obtained from 22 additional patients Results We identified significantly differentially expressed lncRNAs(119899 = 3146) and mRNAs (119899 = 2208) in SSCC relative to noncancerous tissues The GO annotation indicated that there are somecore gene products that may be attributed to the progress of SSCCThe pathway analysis identified many pathways associated withcancerThe results of lncRNA-mRNAnetwork and gene signal-network implied some core lncRNAsmRNAsmight play importantroles in SSCC pathogenesis The results of qRT-PCR showed that all of the 5 lncRNAs were differentially expressed and consistentwith the microarray results Conclusion Our study is the first screening and analysis of lncRNAs expression profile in SSCC andmay offer new insights into pathogenesis of this disease

1 Introduction

Head and neck squamous cell carcinoma is the sixth mostcommon malignancy worldwide Sinonasal squamous cellcarcinomas (SSCC) are rare tumor estimated to account forapproximately 3ndash6 of all head and neck squamous cellcarcinomas SSCC originate in the respiratory epithelium ofthe sinonasal cavities [1] Approximately 60of SSCC arise inthe maxillary sinus 20ndash30 in the nasal cavity 10ndash15 in theethmoid sinuses and sim1 in the frontal and sphenoid sinuses[2 3] Environmental factors such as wood dust and textilemay play a critical role in the development of SSCC As inother head and neck squamous cell carcinomas smoking is aknown risk factor [4] Males who have greater occupationalexposure to carcinogens are affected twice as often as femalesIt is controversial that chronic inflammatory sinus diseasecould influence development of SSCC Human papilloma

virus (HPV) types 16 and 18 may be implicated in malignanttransformation of inverted papillomas [5] The low incidenceof SSCC combined with their nonspecific symptoms oftenleads to a critical delay in diagnosis Treatment for SSCCis usually primarily surgical with adjuvant radiotherapy andsometimes with adjuvant chemotherapy for all except smalltumors [1] Surgical management of SSCC is of great chal-lenge due to its anatomical complexity especially advancedSSCC involving eye skull base or infratemporal fossa Inspite of major advances in the therapy of SSCC includingsurgery and chemoradiotherapymethods the 5-year survivalrate is still very low (30ndash50) [1]

LongnoncodingRNAs (lncRNAs) are a subset of noncod-ing RNAs gt200 nucleotides in length and do not encode anyprotein Due to the poor evolutionary conservation relativeto the protein coding regions of the genome lncRNAs wereonce considered as transcriptional noise or junk and have

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 1095710 10 pageshttpdxdoiorg10115520161095710

2 BioMed Research International

Table 1 Clinical parameters of 6 SSCC patients that underwent lncRNA expression profiling

Specimennumber ofSSCC tissues

Gender Age (years) Exposure factor TNM stage Histologicdifferentiation

Specimen number ofnoncancerous tissues

F5 Male 81 Wood dust T1N0M0 Well G2F9 Male 46 Tobacco smoke T3N0M0 Poorly G10A3 Male 52 Tobacco smoke T4N0M0 Moderately B2D6 Male 72 Wood dust T4N0M0 Moderately F1F2 Male 52 Tobacco smoke T3N0M0 Moderately H2D2 Male 66 Tobacco smoke T3N0M0 Moderately to Poorly D9

not been well studied historically Recently considerable evi-dence has been accumulating showing that aberrant expres-sion of lncRNAs contributes to the development of humancancers [6ndash8] lncRNAs contribute to tumor developmentthrough numerous different cellular processes ranging fromtranscriptional and posttranscriptional regulation of relevantgenes to the control of cell cycle distribution cell differ-entiation and epigenetic modifications lncRNAs may beinvolved in cell proliferation tumor invasion metastasis orapoptosis process lncRNAs are pervasively transcribed andhave a critical role in genome regulation [6 7 9] Howeverto our knowledge little is known about lncRNAs expressionprofile in SSCC and the potential pathways regulating SSCCinvasiveness remain poorly understood

This pilot study aimed to identify aberrantly expressedlncRNAs profile of SSCC and explore their potential func-tionsThis study will help us to understand the tumorigenesisand development of SSCC and provide some new biomarkersthat may be critical to the developmental cascade

2 Materials and Methods

21 Patients and Tissue Samples A total of 28 pairs of pri-mary SSCC tissues and their paired adjacent noncanceroussinonasal tissues were surgically obtained from adult patientsundergoing treatment at Anzhen Hospital and TongrenHospital (two tertiary academic centers in Beijing China)between January 2013 and August 2014 During surgeryfresh tumor tissue and paired noncancerous tissue isolatedfrom at least 2 cm away from the tumor border (sometimescontralateral normal sinonasal mucosa) were collected inthe operating room and processed immediately in liquidnitrogen within 15 minutes and then stored in RNA FixerReagent (Bioteke Beijing China) atminus80∘Cprior to total RNAextraction 6 pairs of tissues underwent microarray analysis(Table 1) and the remaining 22 tissues were used in validationstudies by quantitative real-time polymerase chain reaction(qRT-PCR) Tobacco smoke was the most common exposurefactor about 6667 (46) in the microarray analysis seriesand 6364 (1422) in the PCR validation series Wooddust was the second common exposure factor about 3333(26) in the microarray analysis series and 1818 (422)in the PCR validation series Other exposure factors werechronic sinusitis (322) and leather dust (122) All cases were

reviewed by two ormore independent pathologists and noneof the patients had been previously treated with radiotherapyor chemotherapy All tumor staging was determined accord-ing to the tumor-node-metastasis (TNM) staging criteria ofAmerican Joint Committee on Cancer (AJCC) 2010

The Ethics Committee in Clinical Research of CapitalMedicalUniversity approved this study andwritten informedconsent was provided by all patients

22 Transcript Analysis RNA extraction was carried outusing standard methods (Life Technologies RNA Easy Qia-gen Valencia CA USA) Total RNA was quantified by theNanoDrop ND-2000 (Thermo Scientific) and RNA integritywas assessed using Agilent Bioanalyzer 2100 (Agilent Tech-nologies)

Microarray profiling was conducted with the AgilentHuman lncRNA (4lowast180K Design ID 062918) in this exper-iment and data analysis of the 12 samples has been completedin the laboratory of the KPS Biotechnology Company in Bei-jing China The sample labeling microarray hybridizationand washing were performed based on the manufacturerrsquosstandard protocols Briefly total RNAwas transcribed to dou-ble strand cDNAand then synthesized into cRNA and labeledwith Cyanine-3-CTP The labeled cRNAs were hybridizedonto the microarray After washing the arrays were scannedwith the Agilent Scanner G2505C

Feature Extraction software (version 10711 AgilentTechnologies) was used to analyze array images to obtainraw expression data which was processed using GeneSpringBriefly raw data was normalized with the quantile algorithmProbes which had at least 1 out of 2 conditions having75 flags in ldquo119901rdquo were selected for further data analysisDifferentially expressed gene transcripts were later identifiedWe set a standard threshold set for up- and downregulatedgenes of a fold change ge 20 and a 119901 value le 005

Hierarchical clustering was performed to display expres-sion patterns among samples Briefly we calculated thedistance matrix between the gene expression data Oncethis matrix of distances was computed clustering beginsAgglomerative hierarchical processing consisted of repeatedcycles where the two closest remaining items (those with thesmallest distance) are joined by a nodebranch of a tree withthe length of the branch set to the distance between the joineditems The two joined items were removed from the list of

BioMed Research International 3

Table 2 The primer sequences in the present study

lncRNA name Forward primers (51015840-31015840) Reverse primers (51015840-31015840) Amplicon size (bp)NONHSAT066780 GGAACCAGCTACTCCACACC CCTACCCAGGCCAAGTTCTG 182TCONS l2 00030809 TGAAAAACCACAGGCCCACT ACAAACATGTCTCTCATCAGCAC 78NONHSAT125629 GATTTGAATCGGTCGGCGG AGGCATTTCCTCTCACGCC 284NONHSAG040260 ATGCCCTACGAATGTGGACC TCGGCCCACTGCTAAACATC 218NONHSAG043195 GGGAAGGCTGCCTATGAAGG AATTCGGGGTTGCAGGTTCT 184

items being processed and replaced by an item that representsthe new branch The distances between this new item and allother remaining items were computed and the process wasrepeated until only one item remained

23 lncRNA-mRNA Coexpression Networks 119877 function cortest (a test for associationcorrelation between paired sam-ples) was utilized to compute Pearsonrsquos correlation coefficientto measure the gene coexpression The lncRNAsmRNAs(Pearson correlation coefficients ge093) were selected to drawthe network with Cytoscape

According to these data we built lncRNA-mRNA net-work using the correlation coefficients to examine interac-tions between lncRNA and mRNA The value of ldquodegreerdquoin coexpression network indicated that one mRNAlncRNAmight be correlated with several lncRNAsmRNAs

24 GO Analysis and KEGG Pathway Analysis GO analysiswas applied to analyze the main function of the differentialexpression genes according to the GO database Pathwayanalysis was used to find out the significant pathway of thedifferential genes according to KEGGWe used Fisherrsquos exacttest and 1205942 tests to select the significant pathway and thethreshold of significance was defined by 119901 value and falsediscovery rate (FDR) The enrichment Re was calculatedusing standard methods with a 119901 value (hypergeometric-119901value) denoting the significance of the pathway correlatedwith the conditions with a threshold of 119901 lt 005 adjustedfor multiple comparisons

25 Gene Signal-Network Gene-gene interaction networkwas constructed based on the data of differentially expressedgenes Java was utilized to build and analyze molecularnetworks After parsing the whole KEGG database selectedgenes involved in relevant pathways were extracted and thestudy pathway network was generated with the help of thepathway topology in the KEGG database

26 qRT-PCR Analysis Total RNA was extracted and puri-fied using standard methods (Life Technologies RNA EasyQiagen Valencia CA USA) M-MLV reverse transcription(Promega) was utilized to synthesize cDNA 5 lncRNAexpressions in sinonasal tissues were measured by qRT-PCRwhich was performed on the ABI 7500 qPCR systemwith theprimer pairs listed in Table 2 The raw quantifications werenormalized to the beta-actin gene values for each sample and

fold changes were shown as mean plusmn SD in three independentexperiments each in triplicate

27 Statistical Analysis All data were expressed as the meanplusmn SD or proportions where appropriate Expression levelsbetween SSCC tissues and adjacent nontumor tissues wereanalyzed by paired-sample 119905-tests119901 valueslt005 (two-tailed)indicated statistical significance The Statistical Program forSocial Sciences (SPSS) 210 software (SPSS Chicago ILUnited States) was employed to perform all of the statisticalanalyses

3 Results

31 Overview of lncRNA Profile Out of a collection of 78243lncRNAs and 32776 mRNAs probes our lncRNA expressionprofile of 6 malignant sinonasal tissue and correspondingnormal tissue samples from patients with SSCC indicateddysregulation of 673 (821 upregulated and 1103 downreg-ulated transcripts) of mRNA and 402 (1174 upregulatedand 1098 downregulated transcripts) of lncRNA transcriptsin SSCC tissues (fold change gt2 119901 lt 005) (Figure 1)As expected the lncRNA and mRNA expression profilesallowed distinguishing malignant and normal tissue samplesaccurately based on the molecular signature

Out of the group of RNAs that were upregulated lncRNANONHSAT096777 and mRNA HORMAD1 showed thegreatest degree of demonstrated upregulation with 212076-and 91757-fold increases respectively of those that weredownregulated lncRNA TCONS l2 00002973 and mRNAANKRD30A demonstrated the greatest degree of downregu-lation with 298204- and 275902-fold decreases respectively(Tables 3 and 4)

Hierarchical clustering of the lncRNAs and mRNAs pro-file was performed using cluster 302 hierarchical clusteringof the expression of the top 100 dysregulated lncRNAs andtop dysregulated 100 mRNAs based on centered Pearson cor-relation clearly separated SSCC tissues from correspondingnormal tissues (Figure 2)

32 lncRNA-mRNA Coexpression Network We constructedthe lncRNA-mRNA coexpression network to identify theinteractions between mRNAs and lncRNAs The resultsshowed that the coexpression network was composed of 787network nodes and 8478 connections between 445 lncRNAsand 342 mRNAs Within this coexpression network 7635


78243Up

1174

Down1098

Excluded874

402Total lncRNAs

Upregulated lnRNAs (screened) 1174Downregulated lncRNAs (screened) 1098 Excluded lncRNAs 874

Total lncRNAs 78243Significantly different expressed lncRNAs 3146 (402)

(a)

32776Up821

Down1103 Excluded

284673

Upregulated mRNAs (screened) 821Downregulated mRNAs (screened) 1103 Excluded mRNAs 284

Total mRNAs 32776

Total mRNAs

Significantly different expressed mRNAs 2208 (673)

(b)

Figure 1 (a) Brief microarray results of lncRNAs Expression levels of 78243 lncRNAs were assessed in 6 pairs of SSCC tissues and pairedadjacent noncancerous sinonasal tissues using Agilent Human lncRNA 4lowast180Kmicroarrays Compared with paired adjacent noncanceroustissues 3146 lncRNAs (402) had significant changes in expression levels (fold change gt2 119901 lt 005) A total of 874 lncRNAs were excludeddue to low expression levels A total of 3146 lncRNAs were then identified from the screen with 1174 upregulated and 1098 downregulated(b) Brief microarray results of mRNAs Expression levels of 32776 mRNAs were assessed in 6 pairs of SSCC tissues and paired adjacentnoncancerous sinonasal tissues using Agilent Human lncRNA 4 lowast 180Kmicroarrays Compared with paired adjacent noncancerous tissues2208 mRNAs (673) had significant changes in expression levels (fold change gt2 119901 lt 005) A total of 284 mRNAs were precluded due tolow expression levels A total of 2208 lncRNAs were then identified from the screen with 821 upregulated and 1103 downregulated

Table 3 Top 20 aberrantly expressed lncRNAs in microarray for 6 pairs of SSCC and paired adjacent noncancerous sinonasal tissues

Probe name 119901 FC (abs) Regulation ncRNA accession Gene symbol ChrCUST 13287 PI429545395 0019137 2393843 Down NONHSAT096777 STATH chr4CUST 21727 PI429545388 0039463 8490894 Down NONHSAT009094 PIGR chr1CUST 76033 PI429545399 0024086 8232558 Down NONHSAT013344 MSMB chr10CUST 5401 PI429545406 0024154 7848107 Down NONHSAG018364 NONHSAG018364 chr16CUST 82017 PI429545376 906119864 minus 04 7664131 Down TCONS l2 00029708 linc-RUSC2 chr9CUST 13257 PI429545395 0019957 7585599 Down NONHSAT096773 STATH chr4CUST 13277 PI429545395 0023234 7490795 Down NONHSAT096776 STATH chr4CUST 30951 PI429545395 949119864 minus 05 6720975 Down NONHSAT100745 CTD-2351A81 chr5CUST 91115 PI429545399 004924 6520921 Down NONHSAT017390 MUC5B chr11CUST 5411 PI429545406 0020191 5605385 Down NONHSAT147874 ZG16B chr16CUST 17568 PI429545406 002086 5366862 Down NONHSAT143547 HP chr16CUST 32391 PI429545395 0001238 5126765 Down NONHSAT101166 C7 chr5CUST 57295 PI429545380 132119864 minus 04 4987698 Down FR352905 FR352905 chr19CUST 15837 PI429545395 774119864 minus 04 4776275 Down NONHSAT097405 MMRN1 chr4CUST 32401 PI429545395 0001893 4153056 Down NONHSAT101168 C7 chr5CUST 58955 PI429545388 103119864 minus 05 4074976 Up NONHSAT075623 HOXD11 chr2CUST 72258 PI429545388 200119864 minus 04 3985786 Down NONHSAT087567 CHL1 chr3CUST 58985 PI429545388 509119864 minus 06 3835 Up NONHSAT075626 HOXD10 chr2CUST 32370 PI429545395 0007453 3321905 Down NONHSAG040260 NONHSAG040260 chr5CUST 24853 PI429545410 0003046 3134336 Down XR 2535191 LOC101928556 NAFC fold change Chr chromosome NA not annotated

pairs connections presented as positive and 843 pairs con-nections presented as negative (Figure 3) This coexpressionnetwork indicated that one lncRNA (NONHSAT041869)could target 118 mRNAsmRNAs at most and one mRNA(EXO1) could correlate with 122 lncRNAsmRNAs at most

The results implied that EXO1 CDCA5 and BUB1Bmay playkey roles in SSCC process and development

33 Function Analysis of Differentially Expressed GenesFunctional roles of lncRNAs can only be indirectly predicted


F2 F5 A3 D6 F9 D2 D9 F1 G10 B2 G2 H2

NONHSAT090879NONHSAT090880NONHSAT090878FR352905NONHSAT066780ENST00000606596NONHSAT073797NONHSAT097405NONHSAT098696NR_1043161XR_2458601NONHSAG011177NONHSAT096141NONHSAG038507XR_2425831XR_2462401NONHSAT091224XR_2436261NONHSAT122727NONHSAG043541NONHSAT123617NONHSAT096795NONHSAT003040NONHSAT100745TCONS_l2_00029708NR_0154171FR039099NR_0282941NR_0282931NONHSAT087567NONHSAG003440NONHSAG032907NONHSAT082262NONHSAT127181NONHSAG044217ENST00000520544NONHSAT029430NONHSAG011671NONHSAT140772NONHSAT136587NONHSAG040260NONHSAT101168NONHSAT101166NONHSAT077036XR_2459271NONHSAT099617NONHSAT099618NONHSAT088643NONHSAT035364NONHSAT017386NONHSAT017383XR_2535191NONHSAT141607NONHSAT077649NONHSAT001331NR_0463711NONHSAT092630NONHSAT092627NONHSAT082326NONHSAT013344XR_2417081NONHSAT017389NONHSAT017390NONHSAT096776NONHSAT096773NONHSAT096777NONHSAT009094NONHSAG018364NONHSAT147874XR_2455391NR_0729791NONHSAT143547NONHSAT122281NONHSAT006326NONHSAT006324TCONS_l2_00008290TCONS_l2_00008291NONHSAT048517NONHSAT029206XR_2413091NONHSAT075626NONHSAT075623NONHSAG050236NONHSAT010554NONHSAT069700NONHSAT119525NONHSAT119526NONHSAG041580NONHSAT104436ENST00000423737ENST00000438810NONHSAG050548NONHSAT027516ENST00000532855NONHSAT083976NONHSAG033487XR_2443771NONHSAG038390NONHSAT097329NONHSAT097328

minus2 minus1 0 1 2

(a)

A3 F5 D6 F2 F9 D2 D9 F1 G10 B2 G2 H2

EGR1GPX3PHYHD1PTGFRFOSBEGR3CXCL12CYP4B1SH3BGRL2CGNL1APODCYP2J2ABCA8PPARGC1ASORBS2PLCB4CRHBPFAM3DSELPSELESLC4A4GSTA5ABI3BPCHRDL1ANGPTL1CHL1C4orf7ODAMBPIFA1MSMBBPIFB1STATHDMBT1C10orf81PIGRMUC5ACMUC7TSPAN8PPP1R1BMPV17LBPIFB3HPRTFF3ZG16BPIPAZGP1CCL28PRR4LPOC6orf58PRB1PRB4EMCNCCL14CRISP2CRISP3TFMLPHSELENBP1DIRAS3PTGER3SORBS1CXCL2PLNC2orf40SCN7ANPY1RC7ADRA2APAX3SYNPO2FAM107AALDH1A2MYH11FOSFMO2LAMB4SERPINB11CACNB4COBLITM2ASTEAP4CTSL2HOXC8HOXC9HOXA10FOXD1HMGA2CYP4F3HORMAD1HOXC10HOXD11TM4SF19ODZ2PCSK9IL36GANLNKRT6AHOXA7SPP1

minus2 minus1 0 1 2

(b)

Figure 2 Heat map of lncRNAs and mRNAs that were often aberrantly expressed in SSCC compared with paired adjacent noncanceroussinonasal tissues (a) Hierarchical clustering analysis of the top 100 dysregulated lncRNAs (b) Hierarchical clustering analysis of the top 100dysregulated mRNAs Each row represents one lncRNA or mRNA and each column represents one tissue sample The relative lncRNA ormRNA expression is depicted according to the color scale Red indicates elevated expression and green indicates reduced expression 2 0 andminus2 are fold changes in the corresponding spectrum Carcinoma group (A3 F5 D6 F2 F9 and D2) Paired adjacent noncancerous group (D9F1 G10 B2 G2 and H2)

by analyzing the functions of their coexpressed mRNAsbecause most lncRNAsrsquo functions have not yet been definedTo investigate underlying biological associations we ran GOand KEGG pathway analysis on the top 500 differentiallyexpressed lncRNAs and mRNAs GO analysis indicated thatthese differentially expressed genes were enriched in 12biological processes the majority were proven to be relatedto cancer-associated biological behaviors the top 3 weremulticellular organismal development mitotic cell cycleand cell cycle The differentially expressed genes also wereenriched in 12 cellular components the top 3 were nucleusextracellular region and cytosol Similarly 12 molecularfunctions were enriched for including protein binding DNAbinding andATP binding KEGG analysis revealed pathwaysassociated with cancer such as microRNAs in cancer p53signaling pathway and PI3K-Akt signaling pathway (Figures4(a)ndash4(d))

34 Gene Signal-Network We performed a signal-net analy-sis to investigate the global network based on the significantlyregulated KEGGWith signal-net we screened the importantdysregulated genes involved in the differences between SSCCand normal tissues (Figure 5) The results showed that thecore genes may have played an important role in SSCCprocess According to the results of this analysis the top 3betweenness genes wereMAPK12 RAPGEF3 and KIT

35 qRT-PCR Validation Five differentially expressed lncR-NAs were randomly selected for validation by means ofqRT-PCR according to themanufacturerrsquos recommendationsNONHSAT125629 and TCONS l2 00030809 were upreg-ulated and NONHSAT066780 NONHSAG040260 andNONHSAG043195 were downregulated in SSCCThe resultsof qRT-PCRwere consistent with those of the microarray All


Figure 3 lncRNA-mRNA coexpression network The SSCC consisted of coexpression relationships between lncRNAs and mRNAs The redcircles denote mRNAs and the blue circles denote lncRNAsThe node degree is indicated by the circle size An edge represents a coexpressionrelationship between mRNA and a lncRNA in the context of SSCC progression

of the 5 lncRNAs were differentially expressed with the sametrend (up- or downregulated) (Figure 6)

4 Discussion

SSCC is a rare disease arising in the epithelium of respi-ratory tract and is very poorly studied from the molecularperspective To date the pathogenesis of SSCC remainsunclear due to its low incidence Only a few studies havefocused on its pathogenesis and potential molecular targetsfor therapy specifically microRNAs [10ndash12] Recently studieshave increasingly shown thatmany types of tumors are closelyassociated with the abnormal expression of lncRNAs [6ndash9]In head and neck cancer tongue cancer [13] laryngeal cancer[14] nasopharyngeal cancer [15] and thyroid cancer [6] areall associated with the abnormal expression of lncRNAsHowever to the best of our knowledge there are no reportson lncRNA expression profiles in SSCC

Here we investigated the lncRNA and mRNA expres-sion profiles of SSCC samples from patients using microar-ray analysis We identified thousands of lncRNAs that areexpressed significantly differently in SSCC compared toadjacent noncancerous tissues including both upregulationand downregulation To some extent false positive resultsdo exist in the microarray detection Therefore 5 lncRNAswere randomly selected to validate the microarray resultsConsistent with the microarray results all of the 5 lncRNAswere differentially expressed based on the results of qRT-PCR

In this study we used GO and KEGG pathway analysesto identify biological functions enriched among the differ-entially expressed mRNAs We found that these mRNAswere involved in a lot of cancer-associated biological pro-cesses cellular components and molecular functions TheGO annotation indicated that these gene products mayaffect the tumorigenesis and development of SSCC The


Mitotic cell cycle

M phase of mitotic cell cycle

Nucleosome assembly

Mitotic prometaphase

Cell division

DNA replication

replicationDNA strand elongation involved in DNA

Mitosis

Cell cycle

G1S transition of mitotic cell cycle

GO biological process

5 10 15 20 250minuslg(p value)

(a)

GO cellular component

Chromosome

Nucleosome

Condensed chromosome kinetochore

Chromosome centromeric region

Nucleoplasm

Kinetochore

Extracellular region

MCM complex

Chromatin

Extracellular matrix


(b)

0 1 2 3 4 5

Microtubule motor activity

Actin binding

Fucosyltransferase activity

Protein binding

Aromatase activity

3-Galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase activity

Sequence-specific DNA bindingtranscription factor activity

Monooxygenase activity

Sequence-specific DNA binding

GO molecular function

minuslg(p value)

Oxidoreductase activity acting on paired donors with incorporation or reduction

(c)

Systemic lupus erythematosus

Cell cycle

DNA replication

Oocyte meiosis

Progesterone-mediated oocyte maturation

Salivary secretion

ECM-receptor interaction

Glycosphingolipid biosynthesis lacto- andneolactoseries

Mismatch repair

Homologous recombination

KEGG pathway

5 10 150minuslg(p value)

(d)

Figure 4 GO and KEGG pathway analysis (a)ndash(c) Top 10 enrichment GO terms for differentially expressed mRNAsThe bar plot shows theenrichment scores (minuslg(119901 value)) of the significant enrichment GO terms (a) GO terms of biological process (BP) (b) GO terms of cellularcomponent (CC) (c) GO terms of molecular function (MF) (d) Top 10 pathways of differentially expressed mRNAs in SSCC The verticalaxis represents the pathway category and the horizontal axis represents the enrichment score (minuslg(119901 value)) of the pathway

KEGG pathway analysis identified that many pathways wererelated to cancer such as microRNAs in cancer P53 sig-naling pathway PI3K-Akt signaling pathway For exampleit has been documented that the p53 signaling pathway isactivated in many solid tumors including SSCC [1 16 17]In the present study p53 signaling pathway was related tolncRNANONHSAT125629This molecule may participate innumerous biological processes including mitotic cell cyclecell division DNA replication G1S transition of mitotic cellcycle and G2M transition of mitotic cell cycle

The global network (gene signal-network) and lncRNA-mRNA network structure analysis were established to showthe core genes that play a critical role in this SSCC genenetwork However how these genes participate in the patho-genesis of SSCC largely remains unknown The analysis

revealed that MAPK12 RAPGEF3 and KIT exhibited themost betweenness centrality and all were related to thecancer progression Thus our preliminary data provide ajustification for the involvement of these genes in SSCCdevelopment For example KIT is associated with variouspathways related to cancer such as pathways in cancer andPI3K-Akt signaling pathway Mutations in this gene areassociated with gastrointestinal stromal tumors [18] lungcancer [19] and breast cancer [20]

The limitation of this study lies in the fact that the samplesize is relatively small Our results require further validationin larger prospective patient cohorts and functional experi-ments both in vitro and in vivo Our results provide somevaluable clues for future function and mechanism studies ofSSCC


Figure 5 Signal-net The interaction network of differentially expressed genes (signal-net) The circles represent important functional genesin SSCC (blue downregulated genes red upregulated genes) the circle size represents the degree of interaction (betweenness centrality) thelines indicate the interactions

5 Conclusions

In summary to our knowledge our study is the first screeningand analysis of lncRNA expression profile in SSCC Theresults show that genes regulated by these lncRNAs are

involved in cancer pathways as a proof of principle Thismay offer new insights into pathogenesis and could be apromising way to dissect the molecular pathogenesis of thisrefractory cancer Our study lays the foundation for furtherinvestigation of this disease Further large scale studies are


Table 4 Top 20 aberrantly expressed mRNAs in microarray for 6 pairs of SSCC and paired adjacent noncancerous sinonasal tissues

Probe name 119901 FC (abs) Regulation Gene symbol Chr Genbank accessionA 33 P3265783 0015708 3796405 Down STATH chr4 NM 003154A 23 P154784 0043979 15944 Down BPIFB1 chr20 NM 033197A 33 P3300312 0034496 1461192 Down DMBT1 chr10 NM 007329A 24 P844984 0030927 1195701 Down PIGR chr1 NM 002644A 23 P86599 0023373 9527439 Down DMBT1 chr10 NM 007329A 24 P146683 0032317 8955136 Down MSMB chr10 NM 002443A 33 P3216570 0003716 7559971 Down MUC5AC NA AJ298317A 23 P218369 392119864 minus 04 7396607 Down CCL14 chr17 NM 032963A 23 P118203 0017284 6804929 Down ZG16B chr16 NM 145252A 21 P0013344 0032196 6189528 Down AZGP1 chr7 NM 001185A 23 P95930 601119864 minus 05 584917 Up HMGA2 chr12 NM 003483A 32 P173662 0002064 5824841 Down CRISP2 chr6 NM 003296A 23 P362694 0036618 5465685 Down C4orf7 chr4 NM 152997A 33 P3245228 0031988 5229204 Down BPIFA1 chr20 NM 130852A 23 P58228 0017304 5018366 Down ODAM chr4 NM 017855A 23 P429998 113119864 minus 04 4827517 Down FOSB chr19 NM 006732A 33 P3233040 0012252 4221305 Down SERPINB11 chr18 NM 080475A 23 P8702 0018377 4033374 Down PIP chr7 NM 002652A 33 P3275702 149119864 minus 05 3386336 Down FMO2 chr1 NM 001460A 21 P0000003 0006532 3100913 Down PRR4 chr12 NM 007244FC fold change Chr chromosome NA not annotated

CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel

Figure 6 qRT-PCR validation qRT-PCR verification of 5 candidatelncRNAs in 22 pairs of SSCC tissues The 119910-axis represents therelative expression levels of lncRNAs Paired 119905-tests (2-tailed) wereperformed to compare the expression levels between carcinoma(C) and noncancerous tissues (N) and a 119901 value lt005 indicatedstatistical significance

warranted to provide convincing evidence for clarifying thefunctions of lncRNAs in SSCC and determining whether

these lncRNAs can serve as new diagnostic biomarkersprognostic factors for survival and therapeutic targets inSSCC

Abbreviations

lncRNA Long noncoding RNASSCC Sinonasal squamous cell carcinomaqRT-PCR Quantitative real-time polymerase chain

reactionGO Gene ontologyKEGG Kyoto Encyclopedia of Genes and

GenomesTNM Tumor-node-metastasisAJCC American Joint Committee on CancerFC Fold changeChr ChromosomeNA Not annotated

Disclosure

The present address of Ju-gao Fang is Dongjiaominxiang1st Dongcheng District Beijing 100730 China The presentaddress of Yong-xiang Wei is 2nd Anzhen Road ChaoyangDistrict Beijing 100029 China

Competing Interests

The authors declare that they have no conflict of interests


Acknowledgments

The authors gratefully acknowledge the assistance of JayantM Pinto MD Associate Professor Section of Otolaryngol-ogy-Head and Neck Surgery Department of Surgery TheUniversity of Chicago who provided useful intellectual inputThis work was supported by Beijing Municipal Adminis-tration of Hospitals Clinical Medicine Development SpecialFunding (Grant no 201311)

References

[1] C Garcıa-Inclan A Lopez-Hernandez M Alonso-Guervos etal ldquoEstablishment and genetic characterization of six uniquetumor cell lines as preclinical models for sinonasal squamouscell carcinomardquo Scientific Reports vol 4 article 4925 2014

[2] J H Turner and D D Reh ldquoIncidence and survival in patientswith sinonasal cancer a historical analysis of population-baseddatardquo Head and Neck vol 34 no 6 pp 877ndash885 2012

[3] B Ansa M Goodman K Ward et al ldquoParanasal sinussquamous cell carcinoma incidence and survival based onsurveillance epidemiology and end results data 1973 to 2009rdquoCancer vol 119 no 14 pp 2602ndash2610 2013

[4] K Zhu R S Levine E A Brann H I Hall L S Caplan and DR Gnepp ldquoCase-control study evaluating the homogeneity andheterogeneity of risk factors between sinonasal and nasopha-ryngeal cancersrdquo International Journal of Cancer vol 99 no 1pp 119ndash123 2002

[5] L Alos S Moyano A Nadal et al ldquoHuman papillomavirusesare identified in a subgroup of sinonasal squamous cell carcino-mas with favorable outcomerdquo Cancer vol 115 no 12 pp 2701ndash2709 2009

[6] X Lan H Zhang Z Wang et al ldquoGenome-wide analysis oflong noncoding RNA expression profile in papillary thyroidcarcinomardquo Gene vol 569 no 1 pp 109ndash117 2015

[7] C Chen Z Li Y Yang T Xiang W Song and S LiuldquoMicroarray expression profiling of dysregulated long non-coding RNAs in triple-negative breast cancerrdquo Cancer BiologyampTherapy vol 16 no 6 pp 856ndash865 2015

[8] Q-Q Yang and Y-F Deng ldquoGenome-wide analysis of longnon-coding RNA in primary nasopharyngeal carcinoma bymicroarrayrdquo Histopathology vol 66 no 7 pp 1022ndash1030 2015

[9] X Shen B Xie Z Ma et al ldquoIdentification of novel long non-coding RNAs in triple-negative breast cancerrdquo Oncotarget vol6 no 25 pp 21730ndash21739 2015

[10] N Nohata T Hanazawa N Kikkawa et al ldquoIdentificationof novel molecular targets regulated by tumor suppressivemiR-1miR-133a in maxillary sinus squamous cell carcinomardquoInternational Journal of Oncology vol 39 no 5 pp 1099ndash11072011

[11] N Nohata T Hanazawa N Kikkawa et al ldquoTumour sup-pressive microRNA-874 regulates novel cancer networks inmaxillary sinus squamous cell carcinomardquo British Journal ofCancer vol 105 no 6 pp 833ndash841 2011

[12] T Kinoshita N Nohata H Yoshino et al ldquoTumor suppressivemicroRNA-375 regulates lactate dehydrogenase B in maxillarysinus squamous cell carcinomardquo International Journal of Oncol-ogy vol 40 no 1 pp 185ndash193 2012

[13] W Gao J Y-W Chan and T-S Wong ldquoLong non-codingRNAderegulation in tongue squamous cell carcinomardquo BioMedResearch International vol 2014 Article ID 405860 2014

[14] Z Shen Q Li H Deng D Lu H Song and J Guo ldquoLong non-coding RNA profiling in laryngeal squamous cell carcinomaand its clinical significance potential biomarkers for LSCCrdquoPLoS ONE vol 9 no 9 Article ID e108237 2014

[15] W Zhang L Wang F Zheng et al ldquoLong noncoding RNAexpression signatures of metastatic nasopharyngeal carcinomaand their prognostic valuerdquo BioMed Research International vol2015 Article ID 618924 13 pages 2015

[16] M-B Franzmann C Buchwald G K Jacobsen and H Lin-deberg ldquoExpression of p53 in normal nasal mucosa and insinonasal papillomas with and without associated carcinomaand the relation to human papillomavirus (HPV)rdquo CancerLetters vol 128 no 2 pp 161ndash164 1998

[17] R Holmila J Bornholdt P Heikkila et al ldquoMutations in TP53tumor suppressor gene in wood dust-related sinonasal cancerrdquoInternational Journal of Cancer vol 127 no 3 pp 578ndash588 2010

[18] S Rossi M Sbaraglia M C DellrsquoOrto et al ldquoConcomitantKITBRAF and PDGFRABRAF mutations are rare events ingastrointestinal stromal tumorsrdquo Oncotarget vol 7 no 21 pp30109ndash30118 2016

[19] L Booth J L Roberts M Tavallai et al ldquoThe afatinib resistanceof in vivo generatedH1975 lung cancer cell clones ismediated bySRCERBB3c-KITc-MET compensatory survival signalingrdquoOncotarget vol 7 no 15 pp 19620ndash19630 2016

[20] J Shao L Gan and J Wang ldquoTransfection of chondromodulinI into human breast cancer cells and its effect on the inhibitionof cancer cell growthrdquo Molecular Medicine Reports vol 13 no5 pp 4303ndash4308 2016

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Clinical parameters of 6 SSCC patients that underwent lncRNA expression profiling

Specimennumber ofSSCC tissues

Gender Age (years) Exposure factor TNM stage Histologicdifferentiation

Specimen number ofnoncancerous tissues

F5 Male 81 Wood dust T1N0M0 Well G2F9 Male 46 Tobacco smoke T3N0M0 Poorly G10A3 Male 52 Tobacco smoke T4N0M0 Moderately B2D6 Male 72 Wood dust T4N0M0 Moderately F1F2 Male 52 Tobacco smoke T3N0M0 Moderately H2D2 Male 66 Tobacco smoke T3N0M0 Moderately to Poorly D9

not been well studied historically Recently considerable evi-dence has been accumulating showing that aberrant expres-sion of lncRNAs contributes to the development of humancancers [6ndash8] lncRNAs contribute to tumor developmentthrough numerous different cellular processes ranging fromtranscriptional and posttranscriptional regulation of relevantgenes to the control of cell cycle distribution cell differ-entiation and epigenetic modifications lncRNAs may beinvolved in cell proliferation tumor invasion metastasis orapoptosis process lncRNAs are pervasively transcribed andhave a critical role in genome regulation [6 7 9] Howeverto our knowledge little is known about lncRNAs expressionprofile in SSCC and the potential pathways regulating SSCCinvasiveness remain poorly understood

This pilot study aimed to identify aberrantly expressedlncRNAs profile of SSCC and explore their potential func-tionsThis study will help us to understand the tumorigenesisand development of SSCC and provide some new biomarkersthat may be critical to the developmental cascade

2 Materials and Methods

21 Patients and Tissue Samples A total of 28 pairs of pri-mary SSCC tissues and their paired adjacent noncanceroussinonasal tissues were surgically obtained from adult patientsundergoing treatment at Anzhen Hospital and TongrenHospital (two tertiary academic centers in Beijing China)between January 2013 and August 2014 During surgeryfresh tumor tissue and paired noncancerous tissue isolatedfrom at least 2 cm away from the tumor border (sometimescontralateral normal sinonasal mucosa) were collected inthe operating room and processed immediately in liquidnitrogen within 15 minutes and then stored in RNA FixerReagent (Bioteke Beijing China) atminus80∘Cprior to total RNAextraction 6 pairs of tissues underwent microarray analysis(Table 1) and the remaining 22 tissues were used in validationstudies by quantitative real-time polymerase chain reaction(qRT-PCR) Tobacco smoke was the most common exposurefactor about 6667 (46) in the microarray analysis seriesand 6364 (1422) in the PCR validation series Wooddust was the second common exposure factor about 3333(26) in the microarray analysis series and 1818 (422)in the PCR validation series Other exposure factors werechronic sinusitis (322) and leather dust (122) All cases were

reviewed by two ormore independent pathologists and noneof the patients had been previously treated with radiotherapyor chemotherapy All tumor staging was determined accord-ing to the tumor-node-metastasis (TNM) staging criteria ofAmerican Joint Committee on Cancer (AJCC) 2010

The Ethics Committee in Clinical Research of CapitalMedicalUniversity approved this study andwritten informedconsent was provided by all patients

22 Transcript Analysis RNA extraction was carried outusing standard methods (Life Technologies RNA Easy Qia-gen Valencia CA USA) Total RNA was quantified by theNanoDrop ND-2000 (Thermo Scientific) and RNA integritywas assessed using Agilent Bioanalyzer 2100 (Agilent Tech-nologies)

Microarray profiling was conducted with the AgilentHuman lncRNA (4lowast180K Design ID 062918) in this exper-iment and data analysis of the 12 samples has been completedin the laboratory of the KPS Biotechnology Company in Bei-jing China The sample labeling microarray hybridizationand washing were performed based on the manufacturerrsquosstandard protocols Briefly total RNAwas transcribed to dou-ble strand cDNAand then synthesized into cRNA and labeledwith Cyanine-3-CTP The labeled cRNAs were hybridizedonto the microarray After washing the arrays were scannedwith the Agilent Scanner G2505C

Feature Extraction software (version 10711 AgilentTechnologies) was used to analyze array images to obtainraw expression data which was processed using GeneSpringBriefly raw data was normalized with the quantile algorithmProbes which had at least 1 out of 2 conditions having75 flags in ldquo119901rdquo were selected for further data analysisDifferentially expressed gene transcripts were later identifiedWe set a standard threshold set for up- and downregulatedgenes of a fold change ge 20 and a 119901 value le 005

Hierarchical clustering was performed to display expres-sion patterns among samples Briefly we calculated thedistance matrix between the gene expression data Oncethis matrix of distances was computed clustering beginsAgglomerative hierarchical processing consisted of repeatedcycles where the two closest remaining items (those with thesmallest distance) are joined by a nodebranch of a tree withthe length of the branch set to the distance between the joineditems The two joined items were removed from the list of












3 Results






78243Up

1174

Down1098

Excluded874

402Total lncRNAs



(a)

32776Up821

Down1103 Excluded

284673


Total mRNAs 32776

Total mRNAs


(b)








F2 F5 A3 D6 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(a)

A3 F5 D6 F2 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(b)








4 Discussion





Mitotic cell cycle


Nucleosome assembly


Cell division

DNA replication


Mitosis

Cell cycle




(a)


Chromosome

Nucleosome



Nucleoplasm

Kinetochore


MCM complex

Chromatin



(b)

0 1 2 3 4 5


Actin binding


Protein binding

Aromatase activity






minuslg(p value)


(c)


Cell cycle

DNA replication

Oocyte meiosis


Salivary secretion



Mismatch repair


KEGG pathway


(d)








5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results






78243Up

1174

Down1098

Excluded874

402Total lncRNAs



(a)

32776Up821

Down1103 Excluded

284673


Total mRNAs 32776

Total mRNAs


(b)








F2 F5 A3 D6 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(a)

A3 F5 D6 F2 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(b)








4 Discussion





Mitotic cell cycle


Nucleosome assembly


Cell division

DNA replication


Mitosis

Cell cycle




(a)


Chromosome

Nucleosome



Nucleoplasm

Kinetochore


MCM complex

Chromatin



(b)

0 1 2 3 4 5


Actin binding


Protein binding

Aromatase activity






minuslg(p value)


(c)


Cell cycle

DNA replication

Oocyte meiosis


Salivary secretion



Mismatch repair


KEGG pathway


(d)








5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















78243Up

1174

Down1098

Excluded874

402Total lncRNAs



(a)

32776Up821

Down1103 Excluded

284673


Total mRNAs 32776

Total mRNAs


(b)








F2 F5 A3 D6 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(a)

A3 F5 D6 F2 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(b)








4 Discussion





Mitotic cell cycle


Nucleosome assembly


Cell division

DNA replication


Mitosis

Cell cycle




(a)


Chromosome

Nucleosome



Nucleoplasm

Kinetochore


MCM complex

Chromatin



(b)

0 1 2 3 4 5


Actin binding


Protein binding

Aromatase activity






minuslg(p value)


(c)


Cell cycle

DNA replication

Oocyte meiosis


Salivary secretion



Mismatch repair


KEGG pathway


(d)








5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















F2 F5 A3 D6 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(a)

A3 F5 D6 F2 F9 D2 D9 F1 G10 B2 G2 H2


minus2 minus1 0 1 2

(b)








4 Discussion





Mitotic cell cycle


Nucleosome assembly


Cell division

DNA replication


Mitosis

Cell cycle




(a)


Chromosome

Nucleosome



Nucleoplasm

Kinetochore


MCM complex

Chromatin



(b)

0 1 2 3 4 5


Actin binding


Protein binding

Aromatase activity






minuslg(p value)


(c)


Cell cycle

DNA replication

Oocyte meiosis


Salivary secretion



Mismatch repair


KEGG pathway


(d)








5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Discussion





Mitotic cell cycle


Nucleosome assembly


Cell division

DNA replication


Mitosis

Cell cycle




(a)


Chromosome

Nucleosome



Nucleoplasm

Kinetochore


MCM complex

Chromatin



(b)

0 1 2 3 4 5


Actin binding


Protein binding

Aromatase activity






minuslg(p value)


(c)


Cell cycle

DNA replication

Oocyte meiosis


Salivary secretion



Mismatch repair


KEGG pathway


(d)








5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mitotic cell cycle


Nucleosome assembly


Cell division

DNA replication


Mitosis

Cell cycle




(a)


Chromosome

Nucleosome



Nucleoplasm

Kinetochore


MCM complex

Chromatin



(b)

0 1 2 3 4 5


Actin binding


Protein binding

Aromatase activity






minuslg(p value)


(c)


Cell cycle

DNA replication

Oocyte meiosis


Salivary secretion



Mismatch repair


KEGG pathway


(d)








5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions






CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















CN

p = 067

p = 000001

p = 002

p = 0122

p = 0012

NO

NH

SAG043195

NO

NH

SAG040260

NO

NH

SAT1

25629

TCO

NS_

l2_0

0030

809

NO

NH

SAT0

66780

0

20

40

60

80

100

Relat

ive e

xpre

ssio

n le

vel




Abbreviations




Disclosure


Competing Interests



Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Aberrant Expression Profile of Long Noncoding RNA in Human ...

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