1417
In oduc ion
P os a e cance (PC) is he second mos equen ly
diagnosed cance ype and he six h leading cause o
cance - ela ed dea hs in males wo ldwide [1]. Localized
PC can be cu ed wi h p os a ec omy and/o adia ion
he apy. Howe e , he e is no cu a i e ea men o
ad anced and cas a ion- esis an disease [2]. The use
o p os a e- speci ic an igen (PSA) o he sc eening o
asymp oma ic men o p os a e cance is known o
educe he disease- speci ic mo ali y, bu sc eening is
associa ed wi h o e diagnosis [3]. The e o e, he e is a
s ong in e es o ind addi ional ma ke s and he apeu ic
a ge s o he diagnosis and ea men o p os a e can-
ce . Mic oRNAs (miRNAs) could se e as such ma ke s
o as d ug a ge s [4].
ORIGINAL RESEARCH
Epigene ically al e ed miR- 193b a ge s cyclin D1 in
p os a e cance
Ki si M. Kaukoniemi1,2,a, Hanna E. Rauhala1,a, Mau o Sca a illi1,2, Leena La onen1,2, Ma i Annala1,
Robe L. Vessella3, Ma i Nyk e 1, Teu o L. J. Tammela4 & Tapio Visako pi1,2
1Ins i u e o Biosciences and Medical Technology - BioMediTech, Uni e si y o Tampe e, Tampe e, Finland
2Fimlab Labo a o ies, Tampe e Uni e si y Hospi al, Tampe e, Finland
3Depa men o U ology, Uni e si y o Washing on, Sea le, Washing on
4Depa men o U ology, Uni e si y o Tampe e and Tampe e Uni e si y Hospi al, Tampe e, Finland
© 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use,
dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Keywo ds
Cyclin D1, mic o-RNA, p os a e cance
Co espondence
Tapio Visako pi, Ins i u e o Biosciences and
Medical Technology, Uni e si y o Tampe e,
Tampe e FI-33014, Finland.
Tel: +358 50 318 5829; Fax: +358 3 364 1291;
E-mail: [email p o ec ed]
Funding In o ma ion
The esea ch leading o hese esul s was
unded by Tampe e Uni e si y Doc o al
P og amme in Biomedicine and
Bio echnology. In addi ion, g an suppo has
been ecei ed om he Sig id Juselius
Founda ion, he Finnish Cul u al Founda ion,
Pi kanmaa Regional und, he Academy o
Finland, he Cance Socie y o Finland, he
Medical Resea ch Fund o Tampe e Uni e si y
Hospi al, and he Eu opean Communi y’s
Se en h F amewo k P og amme P ospeR
(FP7/2007-2013) unde g an ag eemen no.
HEALTH-F2-2007-201438.
Recei ed: 14 Janua y 2015; Re ised: 13 May
2015; Accep ed: 27 May 2015
Cance Medicine
2015; 4(9):1417–1425
doi: 10.1002/cam4.486
aEqually con ibu ing au ho s.
Abs ac
Mic o- RNAs (miRNA) a e impo an egula o s o gene exp ession and o en
di e en ially exp essed in cance and o he diseases. We ha e p e iously shown
ha miR- 193b is hype me hyla ed in p os a e cance (PC) and supp esses cell
g ow h. I has been sugges ed ha miR- 193b a ge s cyclin D1 in se e al
malignancies. He e, ou aim was o de e mine i miR- 193b a ge s cyclin D1
in p os a e cance . Ou da a show ha miR- 193b is commonly me hyla ed in
PC samples compa ed o benign p os a e hype plasia. We ound educed miR-
193b exp ession (P < 0.05) in s age pT3 umo s compa ed o pT2 umo s in
a coho o p os a ec omy specimens. In 22R 1 PC cells wi h low endogenous
miR- 193b exp ession, he o e exp ession o miR- 193b educed CCND1 mRNA
le els and cyclin D1 p o ein le els. In addi ion, he exogenous exp ession o
miR- 193b dec eased he phospho yla ion le el o RB, a a ge o he cyclin
D1- CDK4/6 pa hway. Mo eo e , acco ding o a epo e assay, miR- 193b a ge ed
he 3’UTR o CCND1 in PC cells and he CCND1 ac i i y was escued by
exp essing CCND1 lacking i s 3’UTR. Immunohis ochemical analysis o cyclin
D1 showed ha cas a ion- esis an p os a e cance s ha e signi ican ly
(P = 0.0237) highe exp ession o cyclin D1 compa ed o ho mone- naï e cases.
Fu he mo e, he PC cell lines 22R 1 and VCaP, which exp ess low le els o
miR- 193b and high le els o CCND1, showed signi ican g ow h e a da ion
when ea ed wi h a CDK4/6 inhibi o . In con as , he inhibi o had no e ec
on he g ow h o PC- 3 and DU145 cells wi h high miR- 193b and low CCND1
exp ession. Taken oge he , ou da a demons a e ha miR- 193b a ge s cyclin
D1 in p os a e cance .
Cance
Medicine Open Access
1418 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
K. M. Kaukoniemi e al.miR-193b Ta ge s Cyclin D1 in PC
miRNAs a e small, ~22 nucleo ide long noncoding RNA
molecules ha we e i s disco e ed in Caeno habdi is
elegans [5]. Since hei disco e y, miRNAs ha e been ound
o be impo an egula o s o gene exp ession in o he
o ganisms as well [5, 6]. miRNAs modula e gene exp es-
sion by binding o a complemen a y sequence in he 3’
un ansla ed egion (3’UTR) o a ge messenge RNAs
(mRNAs). The binding o a miRNA leads o he deg ada-
ion o he mRNA molecule o al e na i ely, o he sup-
p ession o mRNA ansla ion, depending on he le el o
complemen a y binding [7]. Because one miRNA can ha e
mul iple a ge s and one mRNA molecule can be a ge ed
by many miRNAs, miRNAs o m a complex le el o egu-
la ion o gene exp ession. In addi ion o gene ic changes
(ch omosomal ea angemen s, dele ions, ampli ica ions,
and mu a ions), epigene ic e en s such as abe an p o-
mo e hype me hyla ion, global hypome hyla ion, and
pos ansc ip ional his one modi ica ion may also cause
he dys egula ion o miRNAs in cance [7]. Se e al miR-
NAs a e de egula ed in p os a e cance and ha e been
shown o a ec apop osis, cell cycle, in acellula signaling,
DNA epai , adhesion/mig a ion, and and ogen signaling
( e iewed in [4]).
We p e iously showed ha hsa- miR- 193b- 3p (aka miR-
193b) may unc ion as an epigene ically egula ed umo
supp esso in p os a e cance [8]. Using bisul i e sequenc-
ing we showed ha miR- 193b is hype me hyla ed in some
PC cell lines, wi h he 22R 1 cell line being he mos
hea ily me hyla ed esul ing in he loss o miR- 193b ex-
p ession in he cells. T ansien ans ec ion o p e- miR-
193b in o 22R 1 cells caused signi ican g ow h educ ion
due o a dec eased ac ion o cells in S- phase o he cell
cycle.
The e a e se e al sugges ed a ge genes o miR- 193b
in di e en cance s, o example, CCND1, ETS1, and
KIT [9–16]. The cyclin D1- encoding gene, CCND1, is
a ge ed by miR- 193b in hepa ocellula ca cinoma [10],
melanoma [11], and panc ea ic cance [14]. Cyclin D1
is equen ly abe an ly exp essed in cance . I egula es
he exp ession o genes ha a e in ol ed in DNA ep-
lica ion and he DNA damage checkpoin . The bes
known unc ion o cyclin D1 is mos likely i s ca aly ic
unc ion as a egula o y pa ne o he cyclin- dependen
kinases (CDKs) 4 and 6. In esponse o cellula s imuli,
cyclin D1 ac i a es CDKs 4 and 6, which in u n phos-
pho yla e a ious p o eins, he p incipal subs a e being
e inoblas oma (RB) p o ein. When phospho yla ed, RB
eleases he E2F ansc ip ion ac o , which ac i a es
genes ha a e necessa y o DNA syn hesis and cell
cycle p og ession om he G1 o S phase. In addi ion
o i s ca aly ic unc ion, cyclin D1 also has nonca aly ic
unc ions, wi h he majo unc ion being ansc ip ional
egula ion [17]. In p os a e cance , cyclin D1 has been
shown o unc ion as a co ep esso o and ogen ecep-
o (AR) [18–20].
Because cyclin D1 is a sugges ed a ge o miR- 193b
in o he cance s, we aimed o s udy whe he i is also a
a ge in p os a e cance . In addi ion, ou goal was o
con i m he hype me hyla ion o miR- 193b in clinical
p os a e cance samples.
Ma e ials and Me hods
Cell lines, xenog a s, and clinical samples
The p os a e cance cell lines 22R 1, PC- 3, LNCaP, and
DU145 we e ob ained om he Ame ican Type Cell
Collec ion (Manassas, VA). LAPC- 4 and VCaP cell lines
we e p o ided by D . Cha les Sawye s (Uni e si y o
Cali o nia a Los Angeles, Los Angeles, CA) and D . Jack
Schalken (Radboud Uni e si y Nijmegen Medical Cen e ,
Nijmegen, he Ne he lands), espec i ely. All cell lines we e
cul u ed unde ecommended condi ions. The 17 PC LuCaP
xenog a s we e p o ided by one o he au ho s (R.L.V.).
All clinical samples we e ob ained om Tampe e
Uni e si y Hospi al (TAUH, Tampe e, Finland). F esh-
ozen issue samples, including 10 benign p os a e
hype plasias (BPH), 26 un ea ed p os a ec omy specimens,
and nine cas a ion- esis an umo s (CRPC), we e used
o s udy miR- 193b me hyla ion, whe eas 78 ho monally
un ea ed PC p os a ec omy specimens (Table S1) we e
used o s udy miR- 193b exp ession. BPH samples we e
ob ained om ansu e h al esec ion (TURP) o (cys o)
p os a ec omies om pa ien s wi h BPH o bladde cance .
Un ea ed cance samples we e ob ained om p os a ec-
omies and CRPC samples om TURP. The samples used
in his s udy we e his ologically examined o con ain >70%
cance ous o hype plas ic issue. Fo cyclin D1 immuno-
his ochemical analysis, a o al o 267 o malin- ixed p os a e
cance specimens (198 om p os a ec omies and 69 CRPC
samples) we e used o cons uc issue mic oa ays (TMAs;
Table S2). E hics Commi ee o Tampe e Uni e si y
Hospi al and Na ional Au ho i y o Medicolegal A ai s
ha e app o ed he use o clinical umo ma e ial.
DNA and RNA ex ac ion om clinical
samples
Fo he me hyla ion analysis, eshly ozen issue blocks
we e cu in o 10 × 20- mic ome e sec ions using a c y-
o ome. DNA was isola ed using an AllP ep RNA/DNA
miniki (Qiagen, Valencia, CA) acco ding o he manu-
ac u e ’s p o ocol. Fo miR- 193b exp ession analysis and
a ays, RNA was isola ed using T izol® eagen (In i ogen,
Li e Technologies Co po a ion, Ca lsbad, CA) acco ding
o he manu ac u e ’s p o ocol.
1419
© 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
miR-193b Ta ge s Cyclin D1 in PCK. M. Kaukoniemi e al.
Mic oa ays
miRNA and mRNA exp ession da a om cell lines was
p oduced using Agilen Tehcnologies’ (San a Cla a, CA)
miRNA mic oa ay sys em ( e sion 1 a ay con aining 470
human and 64 i al miRNAs) [8] and Whole Human Genome
Ki (4 × 44k) (Agilen Tehcnologies, San a Cla a, CA), e-
spec i ely. Fo LuCaP xenog a s A yme ix (San a Cla a,
CA) HG U133 Plus 2.0 mic oa ay was used. The a ays
we e done acco ding o he manu ac u e ’s p o ocols.
Cell ans ec ion and co ans ec ion
To de e mine he e ec o miR- 193b exp ession on cyclin
D1, RB, and pRB le els, 22R 1 cells we e ans ec ed wi h
a inal concen a ion o 5 nmol/L p e- miR- 193b o sc am-
bled con ol using INTERFERin ans ec ion eagen
(Polyplus- ans ec ion, Illki ch, F ance) acco ding o he
manu ac u e ’s ins uc ions. The cells we e collec ed 3 days
a e ans ec ion. miRNA p ecu so s we e pu chased om
Ambion (Applied Biosys ems/Ambion, Aus in, TX).
Fo he Cyclin D1 escue and cell cycle expe imen ,
22R 1 cells we e co ans ec ed wi h 10 μg o plasmid,
pCMV- CCND1 lacking a 3’UTR (Plasmid 19927; Addgene,
Camb idge, MA), o he con ol plasmid pCMV6- AC- GFP
(O iGene Technologies, Rock ille, MD) along wi h ei he
p e- miR- 193b o p e- miR- sc amble a a inal concen a-
ion o 10 nmol/L, using je PRIME ans ec ion eagen
(Polyplus- ans ec ion). The cells we e collec ed 2 days
a e ans ec ion.
Flow cy ome ic analysis
Cells we e ha es ed by ypsiniza ion, washed wi h
phospha e- bu e ed saline (PBS) and ixed in cold 70% E OH.
E hanol was aspi a ed, and he cells we e washed and
ehyd a ed wi h PBS. S aining was pe o med wi h p opidium
iodide (Sigma-Ald ich, S . Louis, MO), and cell cycle
p o iles we e analyzed wi h Accu i C6 Flow cy ome e .
Luci e ase epo e assay
Fo he luci e ase epo e assay, he pSGG- 3UTR plas-
mid was pu chased om Swi chGea Genomics (Menlo
Pa k, CA). The plasmid con ains a luci e ase gene used
wi h he 3’UTR o CCND1. 22R 1 cells we e co ans-
ec ed wi h he 3’UTR plasmid, a con ol plasmid-
con aining Renilla luci e ase and p e- miR- 193b o
p e- miR- sc amble using Lipo ec amine™ 2000 ans ec-
ion eagen (In i ogen) acco ding o he manu ac u e ’s
ins uc ions. Fi e ly and Renilla luci e ase ac i i ies we e
measu ed 24 h a e ans ec ion using he Dual- Glo
Luci e ase Assay Sys em (P omega, Madison, WI). Renilla
luci e ase alues we e used o da a no maliza ion. The
luci e ase assay was pe o med in quad uplica e, and
epea ed ou imes.
Wes e n Blo
Nuclea and cy oplasmic p o eins o RB and pRB wes e n
blo s and o al p o ein o cyclin D1 wes e n blo s we e
isola ed as desc ibed p e iously [21, 22]. A o al o 20 μg
o p o eins we e sepa a ed on 8% (RB, pRB) and 10%
(Cyclin D1) SDS- PAGE gels and blo ed o a PVDF mem-
b ane (Immobilon- P, Millipo e Co p., Bille ica, MA). The
memb anes we e incuba ed wi h p ima y an ibodies agains
phospho- Rb (Se 795, 1:1000, Cell Signaling, Dan e s, MA),
Rb (C- 15: sc- 50, 1:500; San a C uz Bio echnology, Inc., Dallas,
TX) o cyclin D1 (clone SP4, 1:100; Dako, Glos up, Denma k)
and wi h an ibodies agains ib illa in (C13C3, 1:4000; Cell
Signaling) o ac in (pan AB- 5 clone ACTN05, 1:400; Lab
Vision Co p., F emon , CA). A e seconda y an ibody in-
cuba ion (an i- abbi - HRP o phospho- RB, RB, cyclin D1
and ib illa in, and an i- mouse- HRP o Ac in; 1:3000, Dako),
he p o eins we e isualized by au o adiog aphy.
Immunohis ochemis y
An ibodies agains cyclin D1 (dilu ion 1:100, clone SP4;
Dako) and Ki- 67 (dilu ion 1:500, MM1; Leica Biosys ems
Newcas le L d., Newcas le upon Tyne, UK) we e used
wi h a Powe Vision+ Poly- HRP IHC ki (ImmunoVision
Technologies Co., Hillsbo ough, CA) acco ding o he
manu ac u e ’s ins uc ions and as desc ibed by Leinonen
e al. [23]. The slides we e scanned wi h an Ape io
ScanScope XT scanne (Leica Mic osys ems GmbH,
We zla , Ge many). The i ual mic oscope [24, 25] and
he ImmunoRa io web applica ion [26] we e used o sco e
he cyclin D1- s aining in ensi y (0–1 = nega i e and weak,
2 = mode a e, and 3 = s ong) in a blinded ashion.
Quan i a i e eal- ime RT- PCR
Fo miR- 193b q- RT- PCR, a TaqMan mic oRNA Assay
(Applied Biosys ems, Fos e Ci y, CA) and he CFX96
q- RT- PCR de ec ion sys em (Bio- Rad Labo a o ies Inc.,
He cules, CA) we e used acco ding o he manu ac u e s’
ecommenda ions. miRNA exp ession was no malized o
RNU6B exp ession. Fo CCND1 q- RT- PCR, i s - s and
complemen a y DNA syn hesis was pe o med om o al
RNA using AMV e e se ansc ip ase (Finnzymes Inc.,
Espoo, Finland) acco ding o he manu ac u e ’s ins uc-
ions. The exp ession o CCND1 was measu ed wi h Maxima
SYBR G een (Fe men as Inc., Bu ling on, ON, Canada)
and he CFX96 q- RT- PCR de ec ion sys em (Bio- Rad
Labo a o ies Inc.) and was no malized o he
β
- Ac in
1420 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
K. M. Kaukoniemi e al.miR-193b Ta ge s Cyclin D1 in PC
e e ence gene. The p ime sequences used o he CCND1
and β- Ac in q- RT- PCR we e: CyclinD1 o 5’- CCCTCGGTG
GGTCCTACTTCAA- 3’, CyclinD1 e 5’- TGGCATTTTGG
AGAGGAAGT- 3’, and Bac in 4 5’- TGGGACGACATGGAG
AAAAT- 3’, Bac in 4 5’- AGAGGCGTACAGGGATAGCA- 3’.
miR- 193b me hyla ion analysis
Me hyla ed DNA was en iched using Me hylMine ™
Me hyla ed DNA En ichmen Ki (In i ogen) acco ding o
he manu ac u e ’s ins uc ions. B ie ly, 2 μg o genomic
DNA was agmen ed by sonica ion. Me hyla ed DNA was
en iched by binding o magne ic beads coa ed wi h he
me hyl- CpG- binding domain o he human MBD2 p o ein
(Me hyl- CpG Binding Domain P o ein 2) and elu ed as a
single ac ion using 2 M NaCl. Finally, he DNA was
e hanol- p ecipi a ed and esuspended in 50 μL o DNase- ee
wa e . Fo q- PCR, iQTM SYBR® G een supe mix and CFX96
q- RT- PCR de ec ion sys em (Bio- Rad Labo a o ies Inc.) we e
used. The sequences o he p ime s used in he q- PCR we e:
miR- 193b_DMR_ 1 5’- TGGCGTTTCTGG TTTCTCTT- 3’
and miR- 193b_DMR_ 2 5’- CGCACCTTTTCTCCTCAT
TT- 3’. Each sample was un in duplica e. The me hyla ion
s a us o miR- 193b was calcula ed om he elu ion- ac ion
signal in ela ion o he o al q- PCR signal.
G ow h cu es
Fo g ow h analysis wi h he CDK4/6 inhibi o PD0332991
(Selleck Chemicals, Hous on, TX), he 22R 1 cells we e
seeded in 24- well pla es in quad uplica e o each inhibi-
o concen a ion. The ollowing day, he cells we e imaged
(day 0), and hen ea ed wi h 0 nmol/L, 100 nmol/L,
500 nmol/L, o 2000 nmol/L inhibi o . The medium was
eplaced e e y o he day wi h esh medium con aining
he inhibi o . The cells we e imaged daily wi h an Olympus
IX71 mic oscope wi h he OASIS au oma ion con ol sys-
em and Su eyo imaging so wa e e sion 5.5.5.26
(Objec i e Imaging L d., Camb idge, UK). Cell g ow h
was analyzed by measu ing he cell su ace a ea wi h an
in- house mac o and ImageJ so wa e (NIH, Be hesda, MD).
The expe imen s we e epea ed h ee imes.
S a is ical analysis
An unpai ed es was used o calcula e he signi ican
di e ence in cell cycle analysis and in luci e ase ac i i y
be ween he p e- miR- sc amble and p e- miR- 193b ans-
ec ed samples as well as he di e ence in miR- 193b ex-
p ession be ween di e en ially s aged p os a ec omy
umo s. The pai ed es was used o calcula e he P- alue
o he g ow h cu e assays on he las day o he expe i-
men . Fishe ’s exac es was used o assess he signi ican
di e ence in miR- 193b me hyla ion in clinical samples.
Fishe ’s exac , chi- squa e, Mann–Whi ney U, and unpai ed
es s we e used o analyze he associa ion be ween clin-
icopa hologic a iables. A P- alue <0.05 was conside ed
signi ican . Spea man’s co ela ion was used o s udy he
co ela ion o miR- 193b and CCND1 exp ession in cell
lines and xenog a samples in mic oa ay analysis.
Resul s
We ha e p e iously shown ha miR- 193b is s ongly hy-
pe me hyla ed in 22R 1 cells and mode a ely me hyla ed
in VCaP cells bu is no me hyla ed in LAPC- 4, LNCaP,
DU145, PC- 3, EP156T, o P EC cells, as de e mined by
bisul i e sequencing [8]. In addi ion, we sequenced i e
un ea ed and ou CRPC clinical specimens and ound
inc eased me hyla ion [8]. He e, we wan ed o con i m
hype me hyla ion o miR- 193b in clinical specimens. We
ecen ly pe o med genome- wide me hyla ion analysis o
me hyla ion by MeDIP- sequencing (unpubl. da a) and
ound ha he mos di e en ially me hyla ed genomic
egion in p oximi y o miR- 193b is 16:14396975–14397475
(GRCh37), which is loca ed 349 bp ups eam o he miR-
193b gene. He e, we measu ed he me hyla ion o miR-
193b in ha egion in BPH (n = 10), PC (n = 26) and
CRPC (n = 9) samples using Me hylMine - qPCR. The
samples we e classi ied as me hyla ed i hey showed mo e
han 20% me hyla ion. Acco ding o ou analysis, miR-
193b was signi ican ly mo e me hyla ed in cance han
in BPH (P < 0.0001; Fig. 1A).
Nex , we measu ed he exp ession o miR- 193b in a
la ge se (n = 78) o p os a ec omy samples and ound
ha he exp ession was lowe (P < 0.05) in s age pT3
umo s han in s age pT2 umo s (Fig. S1). Howe e , we
did no ind an associa ion be ween miR- 193b exp ession
and he Gleason sco e o p og ession- ee su i al (da a
no shown).
Because CCND1 is a sugges ed a ge o miR- 193b a ge
in hepa ocellula and panc ea ic ca cinoma and melanoma
[10, 11, 14], we aimed o de e mine i i is also a a ge
in p os a e cance . The exp ession le els o miR- 193b and
CCND1 we e ob ained using exp ession mic oa ays o PC
cell lines and xenog a samples. The a ay da a showed a
nega i e co ela ion be ween miR- 193b and CCND1 exp es-
sion in he cell lines (Fig. 1B, = −0.7714) as well as in
he xenog a samples (Fig. 1C, = −0.2522). We hen
s udied he exp ession o CCND1 mRNA and p o ein le els
in 22R 1 cells ansien ly ans ec ed wi h p e- miR- 193b.
A ma ked educ ion in he CCND1 exp ession a bo h he
mRNA (Fig. 2A) and p o ein (Fig. 2B) le el was de ec ed
in cells ans ec ed wi h p e- miR- 193b compa ed o he
sc ambled p e- miR ans ec ed cells. Simila o 22R 1 cells,
he Cyclin D1 p o ein exp ession was diminished also in
1421
© 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
miR-193b Ta ge s Cyclin D1 in PCK. M. Kaukoniemi e al.
VCaP cells ansien ly ans ec ed wi h p e- miR- 193b (Fig.
S2). In addi ion, a ma ked educ ion was de ec ed in phos-
pho- RB p o ein le el in nuclea p o ein ac ions (Fig. 2C).
To con i m ha miR- 193b binds o he 3’UTR - egion
o he CCND1 gene in p os a e cance cells, we pe o med
a luci e ase epo e assay. A pSGG- luci e ase plasmid
con aining CCND1 3’UTR a he 3’ end o he luci e ase
gene was ans ec ed oge he wi h ei he p e- miR- 193b
o p e- miR- sc amble in o 22R 1 cells which exp ess low
le els o miR- 193b endogenously. The inhibi ion o
luci e ase ac i i y was obse ed when he cells we e ans-
ec ed oge he wi h he plasmid and p e- miR- 193b
(P < 0.05) bu no wi h p e- miR- sc amble (Fig. 3A),
con i ming ha miR- 193b a ge s he 3’UTR o CCND1.
To e alua e he signi icance o CCND1 as a miR- 193b
a ge , a escue expe imen was pe o med. We used he
phospho yla ion o RB as a measu e o cyclin D1 ac i i y.
Phospho- RB le els dec eased in cells ans ec ed wi h p e-
miR- 193b. When he cells we e co ans ec ed wi h p e-
miR- 193b and he pCMV- CCND1 plasmid lacking he
3’UTR o CCND1, no such educ ion in phospho- RB le els
was obse ed (Fig. 3B). Simila ly, ansien p e- miR- 193b
ans ec ion caused a educ ion in he numbe o cells
in S and G2/M phase ac ions o cell cycle, whe eas he e
was no di e ence in cells co ans ec ed wi h p e- miR- 193b
and CCND1 lacking 3’UTR (Fig. S3).
Because cyclin D1 egula es he ac i i y o CDK4/6, we
ea ed PC cell lines wi h he CDK4/6 inhibi o PD0332991
a di e en concen a ions and measu ed he e ec on
p os a e cance cell g ow h. 22R 1 and VCaP cells, which
exp ess low le els o miR- 193b and high le els o CCND1,
Figu e 1. miR- 193b is me hyla ed in cance samples and has in e se exp ession pa e n compa ed o CCND1. (A) miR- 193b me hyla ion in clinical
samples (BPH, PC, and CRPC) was assessed by Me hylMine - qPCR. miR- 193b and CCND1 exp essions we e s udied by miRNA and mRNA mic oa ay
(B) in p os a e cance cell lines and (C) xenog a samples. Spea man co ela ion coe iciencies a e gi en. BPH, benign p os a e hype plasias; CRPC,
cas a ion- esis an umo s; PC, p os a e cance .
AB
C
Figu e 2. miR- 193b o e exp ession educes he exp ession o CCND1,
he le el o Cyclin D1 p o ein and phospho yla ion le el o e inoblas oma
(RB). 22R 1 p os a e cance cells we e ans ec ed wi h p e- miR- 193b o
p e- miR- con ol. (A) Exp ession le els o CCND1 mRNA was measu ed
using q- RT- PCR. Wes e n blo analysis was used o de ec p o ein
exp ession o (B) Cyclin D1 om o al p o eins and (C) phospho yla ion
le el o RB p o ein om nuclea p o ein ac ion. Ac in and ib illa in
an ibodies we e used as loading con ols.
A
BC

1422 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
K. M. Kaukoniemi e al.miR-193b Ta ge s Cyclin D1 in PC
showed signi ican g ow h supp ession a a 500 nmol/L
concen a ion (P < 0.05; Fig. 4A, B), whe eas he a ious
concen a ions o he inhibi o had li le e ec on he
g ow h o DU145 (Fig. 4C), LNCaP LAPC- 4 o PC- 3
cells, which exp ess high le els o miR- 193b and low le els
o CCND1 (Fig. S4A–C). Figu e S4D shows ha he ehicle,
dime hylsul oxide (DMSO), has no e ec on he cell g ow h
o 22R 1 cells a 0.4% concen a ion.
To s udy he exp ession o cyclin D1 in clinical samples,
we pe o med an immunohis ochemical analysis on TMAs.
The analysis showed ha he CRPC samples exp essed
highe le els o cyclin D1 compa ed o PC samples
(P = 0.0237; Table 1). In p os a ec omy samples, he cyclin
D1 s aining in ensi y was no associa ed wi h Gleason
sco e, pT s age, o diagnos ic PSA le els. Howe e , he e
was s ong posi i e associa ion be ween cyclin D1 and
he p oli e a ion ma ke Ki- 67 (P < 0.0001) in he p os-
a ec omy coho . CRPC cells exp essing high le els o
cyclin D1 had inc eased Ki- 67 alues, al hough he as-
socia ion was no s a is ically signi ican .
Discussion
He e, we con i med he hype me hyla ion o miR- 193b gene
in p os a e cance . The hype me hyla ion o miR-
193b leads o educed exp ession, as we ha e p e iously
shown [8]. Because miRNAs egula e he exp ession o
p o ein- coding genes, he key ques ion is which p o eins
a e a ge ed by miR- 193b in p os a e cance . CCND1 is a
a ge in hepa ocellula and panc ea ic ca cinoma, as well
as in melanoma [10, 11, 14]. We ha e p e iously shown
ha he o e exp ession o miR- 193b educes he p oli e a-
ion o p os a e cance cells due o a dec eased numbe
o cells in S- phase o he cell cycle [8], sugges ing ha
Figu e 4. CDK 4/6 inhibi o PD0332991 supp esses he g ow h o (A)
22R 1 and (B) VCaP cells bu no (C) DU145 cells. Cells we e ea ed
wi h 0, 100, 500, and 2000 nmol/L concen a ions o he inhibi o and
g ow h was ollowed o 5 o 6 days. Each concen a ion was done in
quad uplica es and each expe imen was done in iplica es, a e ages
om expe imen s ±SEM a e shown. P- alues o g ow h di e ences
be ween di e en concen a ions on day 5 o 6 we e calcula ed using
pai ed - es , *P- alue <0.05, **P- alue <0.01.
A
B
C
Figu e 3. miR- 193b a ge s CCND1 3’UTR. (A) Luci e ase expe imen was
pe o med in 22R 1 cells co ans ec ed wi h pSGG- plasmid con aining
CCND1 3’UTR, Renilla luci e ase plasmid, and p e- miR- 193b o p e- miR-
con ol. Values we e no malized agains Renilla luci e ase ac i i y. The
means o ou expe imen s ±SEM a e shown. *P- alue <0.05 (B) Wes e n
blo analysis o pRB in 22R 1 cells ans ec ed wi h pCMV- CCND1
plasmid lacking CCND1 3’UTR oge he wi h miR- sc amble o miR- 193b.
Fib illa in an ibody was used as loading con ol o nuclea p o eins.
A
B
1423
© 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
miR-193b Ta ge s Cyclin D1 in PCK. M. Kaukoniemi e al.
CCND1 could also be a a ge o miR- 193b in p os a e
cance .
We showed ha he exp essions o miR- 193b and CCND1
a e in e sely co ela ed in p os a e cance cell lines and
xenog a s. Subsequen ly, we demons a ed educed mRNA
and p o ein exp ession o CCND1 in 22R 1 cells an-
sien ly ans ec ed wi h p e- miR- 193b. Using a epo e
assay, we con i med ha miR- 193b a ge s he 3’UTR o
he CCND1 gene in 22R 1 p os a e cance cells.
Conco dan ly, Chen e al. [11]. and Xu e al. [10]. ha e
p e iously shown by luci e ase assay ha miR- 193b a ge s
he CCND1 3’UTR in Malme- 3M malignan melanoma
and HepG2 hepa ocellula ca cinoma cells. In addi ion,
we pe o med a escue expe imen , in which we co ans-
ec ed miR- 193b in o 22R 1 cells wi h and wi hou he
pCMV- CCND1 plasmid lacking he 3’UTR o CCND1.
These esul s show ha CCND1 is a bona ide a ge o
miR- 193b in p os a e cance cells.
To assess he ele ance o cyclin D1 a ge ing by miR-
193b in p os a e cance cells, we s udied he ac i i y o
he cyclinD1–RB pa hway in he egula ion o he G1/S
ansi ion in he cell cycle. The ans ec ion o 22R 1
cells wi h miR- 193b educed he le el o phospho- RB, in
acco dance wi h he known unc ion o cyclin D1 in he
egula ion o RB phospho yla ion. In addi ion, he phos-
pho yla ed RB p o ein le els we e escued in pCMV-
CCND1/miR- 193b cells o hose o he con ol cells. Finally,
when p os a e cance cell lines we e ea ed wi h he
CDK4/6 inhibi o PD0332991, he cell lines esponded o
he ea men acco ding o miR- 193b exp ession/me hyla-
ion s a us and cyclin D1 le els. Those wi h low miR- 193b
and high cyclin D1 (22R 1, VCaP) esponded o he d ug
wi h g ow h inhibi ion, while he o he s did no . These
esul s demons a e ha he down egula ion o cyclin D1
by miR- 193b is unc ionally ele an o p os a e cance
cell g ow h.
To assess he clinical signi icance o cyclin D1, we s ained
TMAs and ound ha he exp ession o cyclin D1 is highe
in CRPC (n = 69) compa ed o ho mone- naï e PC
(n = 198). P e iously, D obnjak e al. [27]. showed in-
c eased cyclin D1 s aining in 22 CRPC bone me as ases
compa ed o 86 p ima y PC umo s. In ou p os a ec omy
samples, he exp ession was s ongly associa ed wi h p o-
li e a ion bu no wi h Gleason sco e, pa hological s a us,
PSA alue, o age a diagnosis. This is in line wi h p e i-
ous s udies showing ha cyclin D1 exp ession is associa ed
wi h he p oli e a ion ma ke Ki- 67 bu no wi h o he
clinicopa hological a iables [27–31].
Because i is known ha one gene can be a ge ed by
se e al miRNAs and ha cyclin D1 is a key egula o o
he cell cycle G1/S ansi ion, i is no su p ising ha
CCND1 has been sugges ed o be a a ge o mo e han
one miRNA in p os a e cance . Bonci e al. epo ed ha
miR15- a and 16- 1 in e ac di ec ly wi h he 3’UTR o
CCND1 and educe cyclin D1 p o ein exp ession, he eby
educing p os a e cell p oli e a ion [32]. Howe e , he
unc ion o CCND1 a ge ing by hese h ee miRNAs is
al e ed by di e en mechanisms in p os a e cance . miR-
193b is epigene ically egula ed and silenced by me hyla ion
[8], whe eas he unc ion o miRs 15- a and 16- 1 is dis-
up ed by dele ions in he encoding ch omosomal egion
13q14 [32]. We ha e p e iously epo ed a homozygous
dele ion o he miR15- a and 16- 1 locus in p os a e cance ,
al hough he equency is ela i ely low [33].
In addi ion o CCND1, he e a e se e al o he sugges ed
a ge s o miR- 193b, such as YWHAZ, PLAU (aka uPA),
and KIT in di e en cance ypes [9–16]. Xie e al. [34].
showed ha he knockdown o cys ic ib osis ansmem-
b ane conduc ance egula o (CFTR) led o he supp ession
o miR- 193b exp ession. They also showed ha he o ced
o e exp ession o miR- 193b comple ely ab oga ed ele a ed
u okinase- ype plasminogen ac i a o (uPA) ac i i y a e
CFTR- knockdown in PC- 3 cells, sugges ing ha he umo -
supp essing e ec o CFTR is media ed h ough he miR-
193b- uPA axis. Howe e , i should be no ed ha miR- 193b
exp ession is low in 22R 1 and VCaP cells [8], which
do no exp ess uPA [35, 36], sugges ing ha uPA is no
he majo a ge o miR- 193b in p os a e cance .
In conclusions, we ha e demons a ed ha he o e -
exp ession o cyclin D1 in p os a e cance is d i en, a
leas pa ly, by he educed exp ession o miR- 193b. The
mechanism o miR- 193b supp ession is he
Table 1. Associa ion o clinicopa hological a iables wi h Cyclin D1
exp ession.
Va iable
Cyclin D1 exp ession
P
Nega i e
(0/1)
Posi i e
(2–3)
P os a ec omy specimens, n (%) 74 (37) 124 (63)
Locally ecu en CRPCs, n (%)120 (29) 49 (71) 0.0237
P os a ec omy specimens
Gleason sco e, n (%)1
<7 24 (33) 46 (37)
7 38 (52) 60 (48)
>7 11 (15) 19 (15) 0.8337
pT S age, n (%)2
pT2 54 (75) 88 (72)
pT3 18 (25) 35 (28) 0.6216
PSA ng/mL (mean ± SD)320.0 ± 31.5 14.3 ± 11.3 0.9786
Age (mean ± SD)462.6 ± 5.2 63.2 ± 4.9 0.4167
Ki- 67 (mean ± SD) 7.1 ± 7.0 13.6 ± 14.5 <0.0001
Locally ecu en CRPCs, n (%)3
Ki- 67 (mean ± SD) 13.2 ± 9.3 20.7 ± 15.8 0.0950
1Chi- squa e es .
2Fishe ’s exac es .
3Mann–Whi ney U- es .
4Unpai ed es .
1424 © 2015 The Au ho s. Cance Medicine published by John Wiley & Sons L d.
K. M. Kaukoniemi e al.miR-193b Ta ge s Cyclin D1 in PC
hype me hyla ion o he DMR ups eam o he miR- 193b
gene. Addi ional s udies a e wa an ed o ansla e hese
indings o clinical bene i s. Fo example, he es o a ion
o miR- 193b exp ession could heo e ically educe he
p oli e a ion o p os a e cance cells. Al e na i ely, he loss
o miR- 193b exp ession could indica e he sensi i i y o
p os a e cance cells o cyclin D1 inhibi ion.
Acknowledgmen s
We hank P. Ma ikainen and M. Vakku i o he skill ul
echnical assis ance. The esea ch leading o hese esul s
was unded by Tampe e Uni e si y Doc o al P og amme
in Biomedicine and Bio echnology. In addi ion, g an sup-
po has been ecei ed om he Sig id Juselius Founda ion,
he Finnish Cul u al Founda ion, Pi kanmaa Regional und,
he Academy o Finland, he Cance Socie y o Finland,
he Medical Resea ch Fund o Tampe e Uni e si y Hospi al,
and he Eu opean Communi y’s Se en h F amewo k
P og amme P ospeR (FP7/2007- 2013) unde g an ag ee-
men no. HEALTH- F2- 2007- 201438.
Con lic o In e es
None decla ed.
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Suppo ing In o ma ion
Addi ional suppo ing in o ma ion may be ound in he
online e sion o his a icle:
Table S1. Desc ip ion o clinical samples used o s udy
miR- 193b exp ession.
Table S2. Desc ip ion o clinical samples used in
immunohis ochemis y.
Figu e S1. miR- 193b exp ession in clinical samples.
Figu e S2. miR- 193b o e exp ession educes he exp es-
sion o CCND1 in p o ein le el in VCaP cells.
Figu e S3. CCND1 lacking 3’ UTR is able o escue cell
cycle e ec s o miR- 193b.
Figu e S4. The e ec o CDK 4/6 inhibi o PD0332991
and dissol en dime hylsul oxide (DMSO) on g ow h
o (A) LAPC- 4, (B) LNCaP, (C) PC- 3, and (D) 22R 1
cells.