Long intragenic non-coding RNA lincRNA-p21 suppresses development of human prostate cancer.

TLDR: This invetsigation aimed to study the roles of lincRNA‐p21 in development of human prostate cancer.

Abstract: Objectives Prostate cancer is one of the most frequent malignancies in men, worldwide, although its underlying mechanisms are not fully understood. Long non-coding RNAs participate in development of human cancers. In this invetsigation, we aimed to study the roles of lincRNA-p21 in development of human prostate cancer. Materials and methods Expression of lincRNA-p21 was assessed by real-time PCR in cell lines and in human tissues. Lentivirus carrying sh-lincRNA-p21, lincRNA-p21 or control constructs were used to determine their effects on cell proliferation and apoptosis. A mouse xenograft model was employed to explore the functions of lincRNA-p21 on cancer cell population growth in vivo. Relationships between p53 downstream genes and lincRNA-p21 levels were explored by real-time PCR, western blotting and chromatin immunoprecipitation. Results LincRNA-p21 was found to be down-regulated in human prostate cancer, and low levels of lincRNA-p21 correlated with high disease stage and prediction of poor survival. We further showed that lincRNA-p21 inhibited prostate cancer cell proliferation and colony formation in vitro and reduced rate of prostate cancer cell population growth in vivo. Study of mechanisms involved revealed that lincRNA-p21 promoted apoptosis and induced expression of p53 downstream genes by regulating p53 binding to their promoters. Finally, we showed that expression of p53 downstream genes was reduced in the malignant prostate tissues, which correlated with lincRNA-p21 level. Conclusions Our findings indicated that lincRNA-p21 inhibited development of human prostate cancer partly by regulating p53 downstream gene expression and partly by apoptotic activation.

Full text

Cell Proliferaon. 2017;50:e12318. wileyonlinelibrary.com/journal/cpr 
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hps://doi.org/10.1111/cpr.12318
© 2016 John Wiley & Sons Ltd
Received:8August2016
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Accepted:19October2016
DOI:10.1111/cpr.12318
Abstract
Objecves: Prostatecancerisoneofthemostfrequentmalignanciesinmen,world-
wide,althoughitsunderlyingmechanismsarenotfullyunderstood.Longnon-coding
RNAsparcipateindevelopmentofhumancancers.Inthisinvetsigaon,weaimedto
studytherolesoflincRNA-p21indevelopmentofhumanprostatecancer.
Materials and methods: ExpressionoflincRNA-p21wasassessedbyreal-mePCRin
celllinesandinhumanssues.Lenviruscarryingsh-lincRNA-p21,lincRNA-p21or
controlconstructswereusedtodeterminetheireectsoncellproliferaonandapop-
tosis.AmousexenogramodelwasemployedtoexplorethefunconsoflincRNA-
p21oncancercellpopulaongrowthinvivo.Relaonshipsbetweenp53downstream
genesandlincRNA-p21levelswereexploredbyreal-mePCR,westernblongand
chromanimmunoprecipitaon.
Results: LincRNA-p21wasfoundtobedown-regulatedinhumanprostatecancer,and
lowlevelsoflincRNA-p21correlatedwithhighdiseasestageandprediconofpoor
survival.WefurthershowedthatlincRNA-p21inhibitedprostatecancercellprolifera-
onandcolonyformaoninvitroandreducedrateofprostatecancercellpopulaon
growthinvivo.StudyofmechanismsinvolvedrevealedthatlincRNA-p21promoted
apoptosisandinducedexpressionofp53downstreamgenesbyregulangp53bind-
ingtotheirpromoters.Finally,weshowedthatexpressionofp53downstreamgenes
was reduced in the malignant prostate ssues, which correlated with lincRNA-p21
level.
Conclusions: Our ndings indicated that lincRNA-p21 inhibited development of
human prostate cancer partly by regulang p53 downstream gene expression and
partlybyapoptocacvaon.
DepartmentofUrology,ShanghaiGeneral
Hospital,SchoolofMedicine,ShanghaiJiao
TongUniversity,Shanghai,China
Correspondence
ShujieXiaandDongliangXu,Departmentof
Urology,ShanghaiGeneralHospital,School
ofMedicine,ShanghaiJiaoTongUniversity,
Shanghai,China.
Emails:dongliangxu@21cn.com;
xsjurologist@163.com
Funding informaon
NaonalNaturalScienceFoundaonof
China,Grant/AwardNumber:81300625;
ShanghaiScienceandTechnology
Commiee,Grant/AwardNumber:
13DZ1940602;ShanghaiHospital
DevelopmentCenter,Grant/AwardNumber:
SHDC12014215;ShanghaiMunicipal
CommissionofHealthandFamilyPlanning,
Grant/AwardNumber:20134423
ORIGINAL ARTICLE
Long intragenic non- coding RNA lincRNA- p21 suppresses
development of human prostate cancer
Xiaohai Wang* | Yuan Ruan* | Xingjie Wang | Wei Zhao | Qi Jiang | Chenyi Jiang |
Yuyang Zhao | Yongzhi Xu | Feng Sun | Yiping Zhu | Shujie Xia | Dongliang Xu
1 | INTRODUCTION
Prostate cancer has become themost frequently diagnosed ma-
lignancy in men and the second leading cause ofcancer deaths
among men worldwide.
1
 The involvement of certain genes and
ofchromosomalaberrations in prostatecarcinogenesishas been
suggested.
2,3
However,themolecularmechanismsunderlyingthe
initiation and progression of prostate cancer remain to be fully
understood.
Thematuraonandspreadofhigh-throughputsequencingtech-
nologieshaveenabledincreasinglycomplexanalysesofthe cellular
transcriptome, with the nominaon of numerous novel RNA spe-
cies.
4,5
Amongthese,longnon-codingRNAs(lncRNAs)over200bp
in length havebeen implicated as fundamental actorsin numerous
*Thesetwoauthorscontributedequallytothiswork.

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molecular processes, including cell dierenaon, lineage specic-
ity,neurologicaldisordersandcancer.
6–8
In human prostatecancer,
therstprominentlncRNA,PCA3,wasiniallydescribedasanovel
biomarkerofprostatecancer,
9
andsubsequentlydenedasaprom-
isingurinetestforthisdisease.
10
Similarly,thelncRNAsPCGEM1,
11
SChLAP1,
12
PCAT-1
13,14
andPCAT-3
15
havebeenimplicatedinpros-
tatecancer.
LincRNA-p21 is a p53-regulated long intragenic non-coding
RNAthathasbeenproposedtoactintransviaseveralmechanisms
includingrepressinggenesinthep53transcriponalnetworkand
regulang mRNA translaon and protein stability.
16
 The physio-
logical and pathological funcons oflincRNA-p21weregradually
dened.Forexample,lincRNA-p21hasbeenidenedasaregu-
latorfor the Warburg eectand as avaluabletherapeuc target
forcancer.
17
Inthevascularsystem,lincRNA-p21regulatedneoin-
maformaon,vascularsmoothmusclecellproliferaon,apopto-
sisandatherosclerosisbyenhancingp53acvity.
18
Thefuncons
oflincRNA-p21 in regulang ofstem cell pluripotency werealso
been idened.
19,20
A previousreportshowed thatexosomallin-
cRNA-p21levelsmayhelptoimprovethediagnoscprediconof
themalignantstateforpaentswithprostatecancer.
21
However,
thephysiologicalandpathologicalrolesoflincRNA-p21inthepros-
tatecancerremainunknown.
Here,wedemonstratethatlincRNA-p21suppressesthedevel-
opmentofhumanprostatecancer.LincRNA-p21isdown-regulated
inhumanprostatecancerssues,andlowlincRNA-p21levelpre-
dicts poor survival. Furthermore, we show that lincRNA-p21 in-
hibits prostate cancer growth in vitro and in vivo. LincRNA-p21
inducesapoptosisinprostatecancercells.Inaddion,lincRNA-21
promotesp53enrichmentatthepromotersofitstargetgenesand
thus regulates the expression of p53 downstream proapoptoc
genes. Finally, we show that the down-regulaon of p53 down-
stream genes is associated with lincRNA-p21 in human prostate
cancerssues.
2 | MATERIALS AND METHODS
2.1 | Paents
Two separate cohorts of paents were included in this study. In
cohort 1, radical prostatectomy specimens were collected from
81 paents treated at the Shanghai General Hospital, School of
Medicine,ShanghaiJiaotongUniversity(Shanghai,China),between
2004and2008.Incohort2,radicalprostatectomyspecimenswere
collectedfrom 66 paentstreatedat the sameinstutebetween
2005and2007.Allprostatecancersampleswerehistopathologi-
callyre-evaluatedindependentlybytwopathologistsbeforefurther
analysis. The 15 normal prostate ssues were obtained from pa-
ents (withoutprostate cancer) undergoing surgery.All the sam-
pleswerestoredat−80°Cforuse.Theclinicalparametersofthe
paentsareshowninTables1and2.Awrienformofinformed
consentwasobtainedfromallpaents,andthestudywasapproved
by the Clinical Research Ethics Commiee of Shanghai Jiaotong
University.
2.2 | Quantave real- me PCR
TotalRNAwasextractedfromprostatecancercellsorhumannor-
mal prostate or prostate cancer ssues with TRIzol (Invitrogen).
ThecDNAwassynthesizedfrom1μgoftotalRNAwithOneStep
RT-PCRKit(Takara).Real-mePCRwasperformedwiththeSYBR
Green(Takara)detecon methodonanABI-7500 RT-PCR system
(AppliedBiosystems).Theprimersusedforreal-mePCRarelisted
in Table 3.
2.3 | Western blot
Whole-cell extracts were obtained by lysing cells in TNTE buer
(50mmol/L Tris, pH 7.4, 150mmol/L NaCl, 1mmol/L EDTA,
10mmol/L sodium pyrophosphate, 0.5% Triton X-100, 1mmol/L
sodium vanadate and 25mmol/L sodium uoride) containing
protease inhibitors (5μg/ml PMSF, 0.5μg/ml leupepn, 0.7μg/
ml pepstan and 0.5μg/ml apronin). The detailed Western blot
procedures have been described previously.
17
 The protein sam-
pleswereanalysedusinganbodiesagainstMdm2(Abcam),Puma
TABLE1 Clinicalcharacteriscsofthepaentswithprostate
cancer(cohort1)
Parameters (n=81)
lincRNA- p21
high (n=34)
lincRNA- p21
low (n=47) P value
Tumourstage
pT2(32) 22 10 .0004
pT3a(19) 7 12
pT3b(18) 4 14
pT4(12) 1 11
Gleasongrade
≤3+3(20) 15 5 <.0001
3+4(28) 16 12
4+3(22) 2 20
≥4+4(11) 1 10
Nodalstage
pN0(65) 31 34 .0356
pN+(16) 3 13
PSA
<4(6) 5 1 .0009
4-10(28) 20 8
10-20(30) 10 20
>20(18) 4 14
Reseconmargin
Negave(41) 20 21 .209
Posive(40) 14 26

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WANG ET AL.
(Proteintech), Noxa (Abcam), Bax (Cell Signaling Technology) and
GAPDH(Sigma-Aldrich).
2.4 | Cell culture and retroviral transducon
ThehumanprostatecancercelllinesLNCaP,DU145andPC3were
obtainedfromtheAmerican Type CultureCollecon(ATCC),PTN2
from Sigma and BPH-1 from YRGenge, and they were cultured in
RPMI1640(Invitrogen)supplementedwith10%FBS(Gibco).
Sh-lincRNA-p21 and control shRNA (sh-Ctrl) lenvirus parcles
were purchased from GenePharma.The shRNA sequence targeng
lincRNA-p21 is 5′-GGAGGACACAGGAGAGGCA-3′. Lenvirus ex-
pressing human lincRNA-p21 was generated by subcloning human
lincRNA-p21 cDNA to the pSLIK lenvirus expression system. For
retroviral packaging,293Tcellswere co-transfectedwith the retro-
viralparcles.Fortransducon,LNCaP,PC-3andDU145cellswere
incubatedwithvirus-containingsupernatantinthepresenceof8mg/
mlpolybrene.Aer48hours,infectedcellswereselectedfor72hours
withpuromycin(1.5mg/ml)orhygromycin(150mg/ml).
2.5 | Cell proliferaon and colony formaon assay
Cell proliferaon rate was monitored by CCK-8 Cell Proliferaon/
ViabilityAssayKit(Sigma)accordingtotheguidelines.
Forcolonyformaonassay,prostatecancercellsweresuspended
in 1.5ml complete medium supplemented with 0.45% low melng
pointagarose(Invitrogen).Thecellswereplacedin35mmssuecul-
tureplatescontaining1.5mLcompletemediumandagarose(0.75%)
ontheboomlayer.Theplateswereincubatedat37°Cwith5%CO
2
for2weeks.Cellcolonieswerestainedwith0.005%crystalvioletand
analysedusingamicroscope.
2.6 | Apoptoc assay
Apoptosis was monitored by Fluorescence Acvated Cell Sorter
(FACS).CellswerewashedtwicewithcoldPBSandthenre-suspended
inbindingbuer(BDBiosciences).Then,100mLofthesoluonwas
transferred to a 5mL culture tube and stained with 5mL each of
allophycocyanin-annexin V(BD Biosciences) and50mg/mL propid-
iumiodide(Invitrogen).Thecellsweregentlymixedandincubatedat
roomtemperaturefor15min.Sampleswereanalysedbyowcytom-
etryusinganLSRIIinstrument(Becton-Dickinson).
Theinduconofapoptosiswasalsomonitoredbyterminaldeoxy-
nucleodyl transferase-mediated dUTP nick end labelling (TUNEL)
method.TheTUNELassaywasperformedaccordingtotheguidelines
recommendedintheTUNELApoptosisKit(R&DSYSTEMS).
2.7 | Tumour xenogra experiments
EqualnumbersofLNCaPcellsstablyexpressingeithercontrolorlin-
cRNA-p21knockdownvectors(5×10
6
)in100μLofa1:1mixtureof
culturemediumandgrowthfactor–reducedMatrigelwereimplanted
subcutaneouslyintotheforelegsof5-week-oldmaleBALB/cathymic
nu/numice (Vital River).Whenthe tumoursreachedapproximately
TABLE2 Clinicalcharacteriscsofthepaentswithprostate
cancer(cohort2)
Parameters
(n=66)
lincRNA- p21
high (n=32)
lincRNA- p21
low (n=34) p value
Tumourstage
pT2(27) 20 7 .0032
pT3a(17) 7 10
pT3b(14) 4 10
pT4(8) 1 7
Gleasongrade
≤3+3(21) 15 6 .0275
3+4(22) 11 11
4+3(20) 5 15
≥4+4(3) 1 2
Nodalstage
pN0(53) 29 24 .0408
pN+(13) 3 10
PSA
<4(5) 4 1 .0092
4-10(20) 14 6
10-20(32) 13 19
>20(9) 1 8
Reseconmargin
Negave(41) 20 21 .2021
Posive(35) 12 23
TABLE3 Primersusedforreal-mePCR
Name Sense (5′- 3′) Ansense (5′- 3′)
lincRNA-p21 CCTGTCCCACTCGCTTTC GGAACTGGAGACGGAATGTC
Mdm2 TCGTCGGGTGAGGGTACTG AACCACTTCTTGGAACCAGGT
Bax CATATAACCCCGTCAACGCAG GCAGCCGCCACAAACATAC
Puma GCCAGATTTGTGAGACAAGAGG CAGGCACCTAATTGGGCTC
Noxa CCAAGCCGTGACCAAGGAC CGCCACATTGTGTAGCACCT
β-acn GACCTGACTGACTACCTCATGAAG GTCACACTTCATGATGGAGTTGAAGG

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8–10mmindiameter,theywerepreparedtoformabreiandthenin-
jectedsubcutaneouslyintonudemice.Tumourgrowthwasmonitored
usingcaliperseach3daysorbytumourweightattheendofstudy
(4weeksforinvivotumourgrowth).Thestudywasapprovedbythe
AnimalResearchEthicsCommieeofShanghaiJiaotongUniversity.
The methods were carried out in accordance with the approved
guidelines.
2.8 | Chroman immunoprecipitaon (ChIP) assay
Cellswere cross-linkedby addingformaldehyde toa nalconcen-
traonof 1%at roomtemperature for10min. Aer washing four
meswith20mlPBSin50mlconicaltubes,cellswerescrapedand
swelled in hypotonic swelling buer (25mmol/L HEPES (pH 7.8),
1.5mmol/L MgCl
2
, 10mmol/L KCl, 0.1% NP-40, protease inhibi-
torcocktailfromSigma)andincubatedonicefor10min.Following
centrifugaonat2000rpmfor5min,thenucleiwerelysedinSDS
lysisbuer(1%SDS,10mmol/LEDTAand50mmol/LTris[pH8.1])
andsonicatedwith Branson 150sonicator.Anbodiesagainst p53
(Abcam)andIgG(SantaCruz)wereusedforIP.Real-mePCRwas
carriedoutwith specicprimerstoamplifythep53-bindingregion
of the Mdm2, Puma, Noxa and Bax promoters. The primers were
describedpreviously.
18
2.9 | Stascal analysis
All values are expressed as the mean±SEM. Stascal dierences
betweentwogroupsweredeterminedusingStudent’sttest.One-
way ANOVA was appliedto analyse data that aremore thantwo
groups.ThecorrelaonoflincRNA-p21levelwithpaents’clinico-
pathologicalvariableswasanalysedbythechi-squaretestorFisher’s
exact test. The Kaplan-Meier method was used to esmate over-
allsurvival. Survival dierences according to lincRNA-p21 expres-
sionwereanalysedbythelog-ranktest.Linearregressionanalysis
was used to analyse the relaonship between lincRNA-p21 and
p53 downstream genes. P value of lessthan 0.05was considered
stascally signicant. Stascal analysis was performed using
GraphPadPrism6.
3 | RESULTS
3.1 | LincRNA- p21 is down- regulated in prostate
cancer and predicts survival
ToinvesgatethepotenalroleoflincRNA-p21 in human prostate
cancer, we rst tested the expression paern of lincRNA-p21 in
humanprostatecancer.TherelaveexpressionoflincRNA-p21innor-
malhumanprostatessuesandhumanprostatecancerssueswere
analysed.WefoundthatlincRNA-p21levelwassignicantly down-
regulatedinprostatecancerssuescomparedwithnormalprostate
ssues(Fig.1A).WealsoanalysedtheexpressionoflincRNA-p21in
prostatecancerssuesandmatchedadjacentnormalprostatessues.
TheresultsalsorevealedthatlincRNA-p21inprostatecancerssues
were lower than that in adjacent normal prostate ssues (Fig.1B).
Furthermore,wealsotestedtheexpressionoflincRNA-p21innormal
prostateepithelialcelllinesandprostatecancercelllinesandfound
thelowerleveloflincRNA-p21inprostatecancercelllinesthanthat
innormalprostateepithelialcelllines(Fig.1C).Altogether,thesere-
sultsimplicatethecorrelaonoflincRNA-p21levelwithhumanpros-
tatecancer.
Tofurtherconrmthiscorrelaon,weanalysedtherelaonship
betweenlincRNA-p21levelandpaents’survival.Weanalysedthe
leveloflincRNA-p21in81casesofpaentsanddividedthepaents
tolincRNA-p21highgroup(n=34)andlincRNA-p21lowgroup(n=47)
bythemeanvalueoflincRNA-p21level.Weperformedchi-square
testandFisher’sexacttesttoanalysethecorrelaonbetweenlin-
cRNA-p21 level and clinical parameters. The results showed that
lowlincRNA-p21expressionwasassociatedwithhightumourstage,
GleasongradeandPSA(Table1).Inaddion,weanalysedthecorrela-
onbetweenlincRNA-p21levelandpaents’survival.Thesurvival
analysisshowedthatlowlincRNA-p21levelpredictsbothoveralland
disease-freesurvival(Fig.2A,B).Thesendingswerealsovalidated
FIGURE1 LincRNA-p21isdown-regulatedinprostatecancer.(A)RelativelincRNA-p21levelinnormalprostatetissues(n=15)andprostate
cancertissues(n=81).***P<.001.(B)RelativelincRNA-p21levelinadjacenttissuesandmatchedprostatecancertissues(n=26).**P<.01.(C)
RelativelincRNA-p21levelinnormalprostateepithelialcelllinesandprostatecancercelllines.**P<.01vsPNT2;##P<.01vsBPH-1

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in another separate cohort (Table2 and Fig.2C,D). Therefore, lin-
cRNA-p21mayactasaprognoscfactorforhumanprostatecancer.
3.2 | LincRNA- p21 regulates proliferaon and colony
formaon of prostate cancer cell growth in vitro
ThesignicantcorrelaonbetweenlincRNA-p21andhumanprostate
cancerpromptedustoinvesgatetheroleoflincRNA-p21inhuman
prostatecancer.Tothisend,wersttestedthefunconoflincRNA-
p21incellularproliferaonandcolonyformaon.Weknockeddown
lincRNA-p21 in three prostate cancer cell lines, LNCaP, PC-3 and
DU145cells (Fig.3A) and found that lincRNA-p21 knockdown fa-
cilitatedthe proliferaonrate of prostatecancer cells(Fig.3B–D).
WealsooverexpressedlincRNA-p21inthesethreeprostatecancer
celllines(Fig.3E),andtheresultsshowedthatlincRNA-p21overex-
pressioninhibitedprostatecancercellproliferaon(Fig.3F–H).We
next invesgated whether lincRNA-p21 could aect the capacity
ofcolonyformaonofprostatecancercells.Signicantly,lincRNA-
p21 knockdown promoted cellular colony formaon (Fig.4A–D),
whereaslincRNA-p21overexpressionreducedtheabilityofcolony
formaoninprostatecancercells(Fig.4E–G).Takentogether,these
dataprovideinvitroevidencethatlincRNA-p21inhibitscellularpro-
liferaonandcolonyformaonofhumanprostatecancercells.
3.3 | LincRNA- p21 regulates prostate cancer
development in vivo
TofurtherinvesgatetheeectoflincRNA-p21onprostatecancer
growthin vivo,weperformed xenograexperimentsusing LNCaP
cells with/without lincRNA-p21 stableknockdown. Wefound that
lincRNA-p21 knockdown markedly increased the growth rate of
LNCaP cell in vivofrom 3weeks postLNCaP cell injecon subcu-
taneously (Fig.5A). In addion, we analysed the tumour size and
weight at the end of xenogra experiments. The results showed
thatlincRNA-p21knockdown strictlypromotedtumourgrowth, as
evidencedbyincreasedtumoursizeandweightinthelincRNA-p21
knockdowngroupcomparedwiththecontrol(Fig.5B,C).Inconclu-
sion,lincRNA-p21knockdownpromotesprostatecancercellgrowth
in vivo.
3.4 | LincRNA- p21 regulates apoptosis of prostate
cancer cells
LincRNA-p21 was previously reported to regulate cell survival
and apoptosis, and we wanted to know whether lincRNA-p21
targeted apoptosis in prostate cancer. We overexpressed lin-
cRNA-p21inprostatecancer cellsandfound thatlincRNA-p21
FIGURE2 lincRNA-p21predictspatients’survivalinhumanprostatecancer.(A–B)Incohort1,alltotal81patientsweredividedinto
lincRNA-p21highgroup(n=34)andlincRNA-p21lowgroup(n=47)bythemeanvalueoflincRNA-p21level.TheKaplan-Meiermethodwasused
toestimateoverallsurvival.SurvivaldifferencesaccordingtolincRNA-p21expressionwereanalysedbythelog-ranktest.(A)LincRNA-p21low
levelpredictspooroverallsurvivalinpatientswithprostatecancer.(B)LincRNA-p21lowlevelpredictspoordisease-freesurvivalinpatients
withprostatecancer.(C–D)Incohort2,alltotal66patientsweredividedintolincRNA-p21highgroup(n=32)andlincRNA-p21lowgroup
(n=34)bythemeanvalueoflincRNA-p21level.TheKaplan-Meiermethodwasusedtoestimateoverallsurvival.Survivaldifferencesaccording
tolincRNA-p21expressionwereanalysedbythelog-ranktest.(C)LincRNA-p21lowlevelpredictspooroverallsurvivalinpatientswithprostate
cancer.(D)LincRNA-p21lowlevelpredictspoordisease-freesurvivalinpatientswithprostatecancer