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The handle http://hdl.handle.net/1887/19855 holds various files of this Leiden University dissertation.
Author: Solingen, Coen van
Title: The role of microRNA-126 in vascular homeostasis
Date: 2012-09-26
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IPIGXVSTSVEXIH [MXL +IRI 4YPWIV -- &MS6EH 0EFSVEXSVMIW :IIRIRHEEP 8LI
2IXLIVPERHW 'IPPW [IVI VIGSZIVIH JSV QMR EX VSSQ XIQTIVEXYVI TPEXIH
MRXVMTPMGEXIEX\GIPPWTIV[IPPMR[IPPWTPEXIERHMRGYFEXIHJSVL EX ' *MVI¾] PYGMJIVEWI ERH 6IRMPPE PYGMJIVEWI WMKREPW [IVI QIEWYVIH YWMRK
(YEP0YGMJIVEWI%WWE]6ITSVXIV7]WXIQ 4VSQIKE ERH0YQEX0& )++
&IVXLSPH&YRHSSVE%YWXVEPME 8LIVEXMSSJ½VI¾]6IRMPPEPYGMJIVEWIWMKREPW[EW
GEPGYPEXIH8SIZEPYEXIXLIVIPEXMZIPYGMJIVEWIWMKREPWGM+)R'XLEX[IVIMRGYFEXIH
[MXLERXEKSQMVTVMSVXSXVERWJIGXMSR[IVIEVFMXVEVMP]WIXEX 7XEXMWXMGEPEREP]WMW
7XEXMWXMGEPWMKRM½GERGISJHMJJIVIRGIWMRKIRIERHQM6I\TVIWWMSR[EWTIVJSVQIH
F] QIERW SJ X[SWMHIH 7XYHIRX´W XXIWX EWWYQMRK IUYEP ZEVMERGIW (MJJIVIRGIW
[IVIGSRWMHIVIHXSFIWMKRM½GERX[LIRT
6IWYPXW
)\TVIWWMSR SJ GIPP EHLIWMSR QSPIGYPIW MR VIWTSRWI XS HMWIEWI
MRHYGXMSR
-RHYGXMSRSJERXM+&1KPSQIVYPSRITLVMXMWMRQMGIEW[IPPEWMRZMZSI\TSWYVI
XS MZ82*Ơ VIWYPXIH MR E VETMH EGXMZEXMSR SJ IRHSXLIPMEP GIPPW EW VITVIWIRXIH
F] E WXVSRK YTVIKYPEXMSR SJ )WIPIGXMR ERH:'%1 Q62% I\TVIWWMSR MR XLI
OMHRI] *MK%¯ERXM+&1*MK&¯82*Ơ -RFSXLQSHIPW)WIPIGXMRTVSXIMR
[EWTVIHSQMRERXP]I\TVIWWIHMRKPSQIVYPEVIRHSXLIPMEPGIPPW[LMPII\TVIWWMSR
SJ:'%1TVSXIMR[EWLMKLIWXMREVXIVMSPIWERHI\TVIWWIHXSEPMQMXIHI\XIRX
MR XLI KPSQIVYPM *MK ' %PWS MR E XLMVH MR¾EQQEXMSR QSHIP MG MRHYGIH F]
LIQSVVLEKMG WLSGO E WMQMPEV TEXXIVR SJ VIWXVMGXIH I\TVIWWMSR SJ )WIPIGXMR MR
KPSQIVYPEV GETMPPEVMIW ERH LMKL:'%1 TVSXIMR I\TVIWWMSR MR EVXIVMSPIW [MXL
GSRGSQMXERX PS[ I\TVIWWMSR MR KPSQIVYPM [EW SFWIVZIH *MK ' 8LMW MQTPMIW
XLEX LIXIVSKIRMG IRHSXLIPMEP VIWTSRWMZIRIWW XS EGYXI MR¾EQQEXSV] WXMQYPM MR
HMJJIVIRX VIREP QMGVSZEWGYPEV WIKQIRXW MW E KIRIVEP VIWTSRWI MVVIWTIGXMZI XLI
REXYVISJXLIWXMQYPYW
)WIPIGXMR ERH :'%1 KIRI I\TVIWWMSR MR VIREP QMGVSZEWGYPEV
WIKQIRXW
8SEREP]^IXLIZEWGYPEVWIKQIRXVIWXVMGXIHIJJIGXWSJMR¾EQQEXSV]WXMQYPMSR
KIRII\TVIWWMSR[IMWSPEXIHKPSQIVYPMERHEVXIVMSPIWJVSQXLIOMHRI]F]PEWIV
QMGVSHMWWIGXMSRTVMSVXSU684'6EREP]WIW8LMWTVSXSGSPGSQFMRIWTVIWIVZEXMSR
SJ62%PIZIPW[MXLTVSZMHMRKMRJSVQEXMSRSRXLISVMKMREPPSGEXMSRSJXLIZEWGYPEV
WIKQIRXW MR XLI OMHRI] 9WMRK XLMW [I GSYPH WLS[ LMKL ERH TVIJIVIRXMEP YT
VIKYPEXMSRSJ)WIPIGXMRQ62%MRKPSQIVYPEVGETMPPEVMIWFSXLMRXLIERXM+&1
QSHIP *MK% ERHMRXLIMZ82*ƠGLEPPIRKIQSHIP *MK& 8SKIXLIV[MXLXLI
TVSXIMRHEXEJVSQ*MKYVIXLMWMRHMGEXIWVIKYPEXMSRSJ)WIPIGXMRI\TVIWWMSRMR
XLIKPSQIVYPEVGSQTEVXQIRXEXXLIXVERWGVMTXMSREPPIZIP7YVTVMWMRKP]:'%1
Q62%PIZIPW[IVIYTVIKYPEXIHXSEWMQMPEVI\XIRXMRFSXLKPSQIVYPMERHEVXIVMSPIW
MRFSXLQSHIPW *MK%ERH& VIZIEPMRKEQEVOIHHMWGSVHERGIMR:'%1KIRI
ZIVWYWTVSXIMRI\TVIWWMSRMRXLIKPSQIVYPMXLEXWYKKIWXIHEQEVOIHVIKYPEXMSRSJ
:'%1I\TVIWWMSREXXLITSWXXVERWGVMTXMSREPPIZIP
*MKYVI)\TVIWWMSRSJ)WIPIGXMRERH:'%1MRXLIOMHRI]
% +IRI I\TVIWWMSR SJ )WIPIGXMR ERH:'%1 MW YTVIKYPEXIH MR XLI OMHRI] EX
XLISRWIXSJERXM+&1KPSQIVYPSRITLVMXMWERH & MRVIWTSRWIXSW]WXIQMG82*Ơ
EHQMRMWXVEXMSR5YERXMXEXMZIKIRII\TVIWWMSRMROMHRI]WEQTPIWSJYRXVIEXIHQMGI
GSRXVSP ERH EX L EJXIV MRHYGXMSR SJ ERXM+&1KPSQIVYPSRITLVMXMW Ơ+&1 VIWTIGXMZIP]82*ƠEHQMRMWXVEXMSRVIPEXMZIXS+%4(,LSYWIOIITMRKKIRI(EXEEVI
TVIWIRXIHEWQIERZEPYIW7(SJERMQEPWTIVXVIEXQIRX T ' -QQY
RSLMWXSGLIQMGEPWXEMRMRKSJ)WIPIGXMR:'%1MROMHRI]GV]SWIGXMSRSJYRXVIEXIH
QMGI GSRXVSP SJERXM+&1KPSQIVYPSRITLVMXMWQMGI ERXM+&1 EJXIVMZEHQMRMWXE
XMSRSJ82*ƠERHEJXIVMRHYGXMSRSJLIQSVVLEKMGWLSGO ,7 7QEPPEVVS[LIEHW
MRHMGEXIXLIPSGEXMSRSJEVXIVMSPIWFMKEVVS[WXLIPSGEXMSRSJKPSQIVYPM
% &
% E-selectin
0.008 0.010 0.012 0.014
*
expression
control
VCAM-1
0.05 0.06 0.07 0.08
*
expression
DGBM
0.000 0.002 0.004 0.006
relative gene
0.00 0.01 0.02 0.03 0.04
relative gene
&
control TNFD
E-selectin
0.003 0.004 0.005
expression
VCAM-1
0.02 0.03
expression
0.000 0.001 0.002
relative gene
0.00 0.01
relative gene
'
E-selectin
control
control anti-GBManti TNFTNFDD HSHS
VCAM-1
QM6PIZIPWSJI\TVIWWMSRJSPPS[XLII\TVIWWMSRTEXXIVRWSJ)K¾
ERH)XW
&EWIHSRXLIVIGIRXHEXETYFPMWLIHF],EVVMWIXEP?AVIKEVHMRKXLIVSPISJ
QM6MRTSWXXVERWPEXMSREPVIKYPEXMSRSJ:'%1I\TVIWWMSR[IL]TSXLIWM^IH
XLEX MR EGYXI MR¾EQQEXMSR XLI SFWIVZIH PMQMXIH KPSQIVYPEV:'%1 TVSXIMR
[EWVIPEXIHXSELMKLI\TVIWWMSRSJQM68SXIWXXLMWL]TSXLIWMW[IEREP]^IH
QM6 PIZIPW MR ZMZS MR EVXIVMSPIW ERH KPSQIVYPM SFXEMRIH F] PEWIV QMGVS
HMWWIGXMSRMR[LMGLXLIIRHSXLIPMEPGIPPMRTYXMRIEGLWEQTPI[EWRSVQEPM^IHYWMRK
XLITERIRHSXLIPMEPQEVOIV'(QM6I\TVIWWMSR[EWWMKRM½GERXP]LMKLIV
MRXLIKPSQIVYPEVWIKQIRXWXLERMRXLIEVXIVMSPEVWIKQIRXW *MK%[LMXIFEVW
8LIWIPIZIPWHMHRSXGLERKIMRVIWTSRWIXSXLIMRHYGXMSRSJEGYXIMR¾EQQEXMSR
*MK%FPEGOFEVW -RGSRXVEWXXLIRSRIRHSXLIPMEPVIWXVMGXIHQMGVS62%QM6
[EWI\TVIWWIHEXWMQMPEVPIZIPWMRXLIX[SVIREPQMGVSZEWGYPEVWIKQIRXWFSXLMR
UYMIWGIRXERHMRMR¾EQQEXSV]GSRHMXMSRW *MK&
&IGEYWIQM6MWERMRXVSRMGTVSHYGXSJXLIKIRI)K¾ ITMHIVQEPKVS[XL
JEGXSVPMOIHSQEMREPWSORS[REWZEWGYPEVIRHSXLIPMEPWXEXMR?A ERHQM6
[EWTVIZMSYWP]VITSVXIHXSJSPPS[IRHSXLIPMEPVIWXVMGXIHI\TVIWWMSRTEXXIVRWSJ
*MKYVI )WIPIGXMR ERH :'%1 KIRI I\TVIWWMSR MR ZMZS EVI
GSQTEVXQIRXEPM^IH MR HMJJIVIRX QMGVSZEWGYPEV WIKQIRXW MR XLI
OMHRI]
&SXLMRERXM+&1KPSQIVYPSRITLVMXMW % ERHMRVIWTSRWIXSW]WXIQMG82*ƠXVIEX
QIRX & KIRI I\TVIWWMSR SJ )WIPIGXMR [EW LMKLP] ERH WTIGM½GEPP] YTVIKYPEXIH
MRKPSQIVYPEVIRHSXLIPMEPGIPPW K [LMPILEVHP]EJJIGXIHMRXLIEVXIVMSPIW E [LMPI
:'%1KIRII\TVIWWMSR[EWMRHYGIHXSEWMQMPEVI\XIRXMREVXIVMSPIW E ERHMR
KPSQIVYPM K 1MGI[IVIWEGVM½GIHEXLEJXIVHMWIEWIMRHYGXMSRERHZEWGYPEVWIK
QIRXW[IVIPEWIVQMGVSHMWWIGXIHTVMSVXSU684'6EREP]WMWEWHIWGVMFIHMR11
(EXEEVITVIWIRXIHEWQIERZEPYIW7(SJERMQEPWTIVXVIEXQIRX KIRII\
TVIWWMSRMRQMGIWYFNIGXIHXSHMWIEWIWXMQYPYW[EWWMKRM½GERXP]HMJJIVIRXGSQTEVIH
XSYRXVIEXIHGSRXVSPQMGIT RHRSXHIXIGXIH
%
&
control
D -GBM control
TNFD
)K¾? A[IEPWSEREP]^IH)K¾I\TVIWWMSRMRXLIX[SQMGVSZEWGYPEVFIHW
8LMW EREP]WMW VIZIEPIH E WMQMPEVP] LMKLIV I\TVIWWMSR SJ )K¾ MR XLI KPSQIVYPEV
GSQTEVXQIRXGSQTEVIHXSMXWI\TVIWWMSRMRXLIEVXIVMSPEVGSQTEVXQIRXFSXL
MRGSRXVSPGSRHMXMSRWERHMRVIWTSRWIXSXLIMR¾EQQEXSV]GLEPPIRKI *MK'
-RGSRXVEWXXSXLISXLIVW)K¾I\TVIWWMSR[EWQSHIWXP]HS[RVIKYPEXIHMRFSXL
GSQTEVXQIRXWYRHIVEGYXIMR¾EQQEXSV]GSRHMXMSRW
;LMPITIVJSVQMRKSYVWXYH],EVVMWIXEPVITSVXIHXLEX)XWMWSRISJXLIQEMR
XVERWGVMTXMSRJEGXSVWMRGSRXVSPSJQM6I\TVIWWMSRMRIRHSXLIPMEPGIPPWMRZMXVS
?A%REP]^MRK)XWI\TVIWWMSRPIZIPWMRXLIX[SZEWGYPEVGSQTEVXQIRXWYRHIV
WXYH]VIZIEPIHEWMKRM½GERXJSPHLMKLIVI\TVIWWMSRPIZIPSJXLMWXVERWGVMTXMSR
JEGXSVMRXLIKPSQIVYPMEWGSQTEVIHXSXLIEVXIVMSPIWFSXLSJ[LMGL[IVIRSX
EJJIGXIHF]XLIMR¾EQQEXSV]TVSGIWW *MK(
QM6 I\TVIWWMSR MW SRP] I\TVIWWIH MR XLI IRHSXLIPMEP GIPPW SJ
KPSQIVYPM
7MRGI PEWIV QMGVSHMWWIGXIH KPSQIVYPM GSRWMWX SJ QSVI GIPP X]TIW FIWMHIW
*MKYVI (MJJIVIRXMEP I\TVIWWMSR SJ QM6 MR VIREP QMGVS
ZEWGYPEXYVI
% &
' (
control D -GBM
IRHSXLIPMEPGIPPW[IEREP]^IHXLII\TVIWWMSRSJQM6QM6)K¾ERH)XW
MRGYPXYVIHKPSQIVYPEVIRHSXLIPMEPGIPPWQIWERKMEPGIPPWERHTSHSG]XIW8LIHEXE
TVIWIRXIHMR*MKYVI%WYTTSVXXLIRSXMSRXLEXQM6I\TVIWWMSRMWIRHSXLIPMEP
GIPP VIWXVMGXIH [MXL HIXIGXEFPI FYX ZIV] PS[ PIZIPW MR QIWERKMEP GIPPW ERH
TSHSG]XIW [LMPI XLI RSRIRHSXLIPMEP VIWXVMGXIH QM6 [EW JSYRH XS FI
I\TVIWWIHEXWMQMPEVPIZIPWMREPPXLVIIKPSQIVYPEVGIPPX]TIW%PWS)K¾I\LMFMXIH
ERIRHSXLIPMEPVIWXVMGXIHI\TVIWWMSRTVS½PI[LMPI)XW[EWI\TVIWWIHXSEWMQMPEV
I\XIRXMREPPXLVIIGIPPX]TIW *MK% 82*ƠQIHMEXIHVIKYPEXMSRSJXLIQM6WERH
KIRIWMRKPSQIVYPEVIRHSXLIPMEPGIPPW[EWSRP]SFWIVZIHJSV)K¾ERH)XW)K¾
[EWHS[RVIKYPEXIHXS SJGSRXVSPPIZIPWEXEPEXIVWXEKISJ82*ƠQIHMEXIH
EGXMZEXMSR L [LMPI)XWI\TVIWWMSRMRGVIEWIHETTVS\JSPHZIVWYWGSRXVSP
[MXLMRLEJXIVWXEVXSJ82*ƠI\TSWYVI *MK&
QM6MWJYRGXMSREPP]EGXMZIMRKPSQIVYPEVIRHSXLIPMEPGIPPW
8SMRZIWXMKEXI[LIXLIVQM6GERFIJYRGXMSREPP]EGXMZIMRXLIKPSQIVYPEV
IRHSXLIPMEP GIPPW [I YWIH VITSVXIV EREP]WMW XS ZEPMHEXI FMRHMRK XS XLI ´986
VIKMSRSJ:'%1 *MK% 4VMSVXSXLIXVERWJIGXMSRI\TIVMQIRXWKPSQIVYPEV
IRHSXLIPMEPGIPPW[IVITVIGYPXYVIHSZIVRMKLX[MXLERYGPISXMHIGLSPIWXIVSP
GSRNYKEXIHVERHSQ62%WIUYIRGI WGVEQFPIQMV SV[MXLMHIRXMGEPP]WM^IHERH
QSHM½IH 62% GSQTPIQIRXEV] XS QEXYVI QM6 ERXEKSQMV ;LMPI MR
WGVEQFPIQMVXVIEXIHGIPPWXLIVIPEXMZI½VI¾]PYGMJIVEWII\TVIWWMSRSJXLIVITSVXIV
TPEWQMH LEVFSVMRK XLI ´986 VIKMSR SJ:'%1 T1-6:'%1 ´986 [EW
PS[ GSQTEVIH XS E GSRXVSP VITSVXIV TPEWQMH PEGOMRK XLIWI WIUYIRGIW T1-6
VITSVX *MK & MRGYFEXMSR [MXL ERXEKSQMV VIWXSVIH PYGMJIVEWI PIZIPW SJ
T1-6:'%1´986[MXLSYXEJJIGXMRKPYGMJIVEWIPIZIPWSJXLIT1-6VITSVXIV
8LIWIVIWYPXWMQTP]XLEXQM6MWJYRGXMSREPMRKPSQIVYPEVIRHSXLIPMEPGIPPWEW
*MKYVI (MJJIVIRXMEP I\TVIWWMSR SJ QM6 MR VIREP QMGVS
ZEWGYPEXYVI
1MGVS62%VIWTIGXMZIP]Q62%I\TVIWWMSRPIZIPWMREVXIVMSPIW E ERHKPSQIVYPEV K GSQTEVXQIRXWMRGSRXVSPQMGI [LMXIFEVW ERHQMGIWYFNIGXIHXSXLIERXM+&1
KPSQIVYPSRITLVMXMW QSHIP FPEGO FEVW 0IZIPW [IVI UYERXM½IH EJXIV PEWIV QMGVS
HMWWIGXMSRSJXLIVIWTIGXMZIQMGVSZEWGYPEVFIHWJVSQJVS^IRQSYWIOMHRI]FMSTWMIW
EWHIWGVMFIHMR1EXIVMEPWERHQIXLSHW8LIPIZIPSJI\TVIWWMSRSJQM6 % [EW WMKRM½GERXP]LMKLIVMRXLIKPSQIVYPEVGSQTEVXQIRXXLERMRXLIEVXIVMSPEVGSQTEVX
QIRX ERH RSX EJJIGXIH F] XLI MR¾EQQEXSV] TVSGIWW [LMPI XLI RSRIRHSXLIPMEP
VIWXVMGXIH QM6 [EW I\TVIWWIH MR FSXL GSQTEVXQIRXW XS E WMQMPEV I\XIRX ERH
EPWSRSXEJJIGXIHF]XLIMR¾EQQEXMSR & 8LII\TVIWWMSRTEXXIVRWSJ)K¾ ' XLI
LSWXKIRISJQM6EW[IPPEWSJ)XW ( EXVERWGVMTXMSRJEGXSVWLS[RXSGSRXVSP
QM6I\TVIWWMSRJSPPS[IHXLIWEQIWTEXMEPI\TVIWWMSRTEXXIVREWSFWIVZIHJSV
QM6;LMPI)K¾[EWQSHIWXP]HIGVIEWIHF]XLIMR¾EQQEXMSRMRHYGIH)XW
[EWRSXEJJIGXIH(EXEEVITVIWIRXIHEWQIERZEPYIW7(SJERMQEPWTIVKVSYT
T EVXIVMSPIWZWKPSQIVYPMT GSRXVSPZWERXM+&1
ERIKEXMZIVIKYPEXSVSJ:'%1I\TVIWWMSR
-R ZMZS WMPIRGMRK SJ QM6 F] ERXEKSQMV VIWYPXIH MR
MRGVIEWIH:'%1TVSXIMRI\TVIWWMSRYTSR82*ƠGLEPPIRKI
8LIEFSZIHEXEWYKKIWXIHXLEXQM6GERTPE]EVSPIMRVITVIWWMRKKPSQIVYPEV
:'%1TVSXIMRI\TVIWWMSRYTSRERMR¾EQQEXSV]GLEPPIRKIERHXLEXXLMWTSWX
XVERWGVMTXMSREPVSPIF]QM6GSYPHI\TPEMRXLISFWIVZIHLIXIVSKIRMG:'%1
TVSXIMR I\TVIWWMSR TEXXIVRW MR XLI QMGVSZEWGYPEXYVI SJ XLI OMHRI]8S WXYH]
[LIXLIV WYGL E VIPEXMSR I\MWXW [I XVIEXIH QMGI [MXL ERXEKSQMV ERH
WGVEQFPIQMV GSRXVSP SPMKSRYGPISXMHIW TVMSV XS ER EGYXI GLEPPIRKI F] MZ
EHQMRMWXVEXMSRSJ82*Ơ%WEGSRXVSPJSVTVSTIVMRHYGXMSRSJMR¾EQQEXMSR[I
JSPPS[IHXLII\TVIWWMSRSJ)WIPIGXMRTVSXIMRF]MQQYRSLMWXSGLIQMWXV] *MK% ERHSFWIVZIHXLEXEWI\TIGXIHXLII\TVIWWMSRSJXLMWTVSXIMR[EWRSXEJJIGXIH
F]ERXEKSQMVSVWGVEQFPIQMVXVIEXQIRX-RGSRXVEWX[ISFWIVZIHEQEVOIH
MRGVIEWIH I\TVIWWMSR SJ:'%1 TVSXIMR MR XLI KPSQIVYPEV GSQTEVXQIRX SJ
*MKYVI)\TVIWWMSRSJQM6QM6)K¾ERH)XWMRKPSQIVYPEV
GIPPWMRZMXVSERHXLIMVVIKYPEXMSRMRKPSQIVYPEVIRHSXLIPMEPGIPPWF]
82*ƠI\TSWYVI
% &EWEP I\TVIWWMSR PIZIPW SJ XLI QMGVS62%W ERH Q62%W [IVI HIXIVQMRIH
EW HIWGVMFIH MR 1EXIVMEPW ERH QIXLSHW F] U684'6 ERH RSVQEPM^IH XS 629
VIWTIGXMZIP] +%4(, & 6IKYPEXMSR SJ XLI I\TVIWWMSR SJ XLI QMGVS62%W ERH Q62%WMRKPSQIVYPEVIRHSXLIPMEPGIPPWI\TSWIHXSRKQ082*ƠJSVLERHL
(EXEEVITVIWIRXIHEWQIERZEPYIW7( R!¯T
1.02.5 3.0 3.5 4.0 4.5 5.0
gene expression miR/RNU48)
0.010 0.015 0.020
gene expression ne/GAPDH)
miR-126 miR-31 0.0
0.5 1.0
relative g (m
Egfl7 Ets1 0.000
0.005 relativeg (ge
al cells Glomerular endothelia Mesangial cells Mesangial cells Mesangial cells Podocytes
%
&
4 5 6
ession U48) 0.03
0.04
*
* ession PDH)
miR-126 miR-31
Egfl7 Ets1
0 4h 24h 0 4h 24h 0.0
0.5 1.03
relative expre (miR/RNU
0 4h 24h 0 4h 24h 0.00
0.01 0.02
relative expre (gene/GAP *
0 4h 24h 0 4h 24h 0 4h 24h 0 4h 24h
XLI QMGI XVIEXIH [MXL XLI ERXEKSQMV *MK 1SVISZIV RSRKPSQIVYPEV
WIKQIRXWEPWSHMWTPE]IHERERXEKSQMVXVIEXQIRXVIPEXIHMRGVIEWIHI\TVIWWMSR
SJ:'%1 TVSXIMR [LMGL MW QSWX PMOIP] HYI XS XLI TVIWIRGI SJ JYRGXMSREP
EPFIMX PS[IV PIZIPW SJ QMV MR XLI SXLIV QMGVSZEWGYPEV WIKQIRXW EW [I
TVIZMSYWP]WLS[IHUYEPMXEXMZIP]F]MRWMXYL]FVMHM^EXMSR?A7GVEQFPIQMVXVIEXQIRX
HMHRSXEJJIGX:'%1TVSXIMRI\TVIWWMSRPIZIPWMRER]SJXLIZEWGYPEVWIKQIRXW
5YERXM½GEXMSR SJ :'%1 TVSXIMR PIZIPW MR [LSPI OMHRI] TVSXIMR MWSPEXIW
F] )0-7% EPWS WLS[IH XLEX:'%1 TVSXIMR PIZIPW [IVI MRGVIEWIH F] XLI
ERXEKSQMVXVIEXQIRX[LMPIWGVEQFPIQMVXVIEXQIRXHMHRSXEJJIGXXLIPIZIPW
*MK&
-R TEVEPPIP [I EREP]^IH XLI QMGVSZEWGYPEV VIWTSRWMZIRIWW XS ERXEKSQMV
EHQMRMWXVEXMSR MR XLI PMZIV [LIVI [I EPWS SFWIVZIH TVSQMRIRXP] IRLERGIH
:'%1 TVSXIMR I\TVIWWMSR MR XLI QMGVSZEWGYPEV WMRYWSMHEP IRHSXLIPMEP GIPPW
[LMGL [EW JYVXLIV WYFWXERXMEXIH F] )0-7% UYERXM½GEXMSR SJ:'%1 TVSXIMR
GSRXIRXSJXLIPMZIV *MK -RGSRXVEWXXLIGETMPPEVMIWSJXLIPYRKWERHLIEVX[IVI
HIZSMHSJWYGLEVIWTSRWI HEXERSXWLS[R 8LMWMQTPMIWXLEXRSXSRP]MRXLI
OMHRI]FYXEPWSMRSXLIVSVKERW]IXRSXMREPPQM6I\IVXWEVSPIMR:'%1
TVSXIMRI\TVIWWMSRGSRXVSPMRVIWTSRWIXSEREGYXIMR¾EQQEXSV]WXMQYPYW
*MKYVI 1M6 MW JYRGXMSREPP] EGXMZI MR KPSQIVYPEV IRHSXLIPMEP
GIPPW
% 7GLIQEXMGVITVIWIRXEXMSRSJT1-6VITSVXERHT1-6:'%1´986TPEWQMHW
&SXLTPEWQMHWGSRXEMRXLI½VI¾]PYGMJIVEWIKIRI-RXLIPEXXIVXLIRXSJ:'%1
´986 [IVI GPSRIH MRXS XLI QYPXMTPI GPSRMRK WMXI HS[RWXVIEQ SJ XLI PYGMJIVEWI
KIRI8LIWIUYIRGIKMZIRFIPS[XLIT1-6:'%1´986MWXLIFMRHMRKWMXIJSV
QM6 & 6IPEXMZI ½VI¾] PYGMJIVEWI I\TVIWWMSR MR KPSQIVYPEV IRHSXLIPMEP GIPPW
TVIMRGYFEXIH[MXLERXEKSQMVERHGSRXVSPWGVEQFPIQMVVIWTIGXMZIP]'IPPW[IVI
XVERWJIGXIH[MXLIMXLIVXLIT1-6VITSVX[LMGLPEGOWXLIQM6GSRWIRWYWSVXLI
T1-6:'%1´986FSXLWGLIQEXMGEPP]WLS[RMRTERIP%(EXEEVITVIWIRXIHEW
QIERZEPYIW7( R! T
Luciferase
SpeI HindIII
pMIR-report
Luciferase
SpeI HindIII
pMIR-VCAM-1 3’UTR
3’UTR VCAM-1
SpeI HindIII
CUGUAUAGUACUGGCAUGGUACG
100
*
ase %)
50 00
relative lucifera expression (%
antagomir-126 scramblemir
antago mir-126
scramblemi r 0
pMIR-report pMIR-VCAM-1 3'UTR
% &
W
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62% ERI[JVSRXMIVMROMHRI]ERHFPSSHTVIWWYVIVIWIEVGL%Q.4L]WMSP6IREP4L]WMSP
**
0MY1/PYKIV17(´%PIWWMS%+EVGME'EVHIRE+ERH4SFIV.76IKYPEXMSRSJ%VXIVMEP:IRSYW
(MJJIVIRGIWMR8YQSV2IGVSWMW*EGXSV6IWTSRWMZIRIWWSJ)RHSXLIPMEP'IPPWF]%REXSQMG'SR
XI\X%Q.4EXLSP
1SPIQE+,IXIVSKIRIMX]MRIRHSXLIPMEPVIWTSRWMZIRIWWXSG]XSOMRIWQSPIGYPEVGEYWIWERH
TLEVQEGSPSKMGEPGSRWIUYIRGIW7IQ8LVSQF,IQSWX
1SPIQE+,IXIVSKIRIMX]MRVIWTSRWIWSJQMGVSZEWGYPEVIRHSXLIPMEPGIPPWHYVMRKMR¾EQQEXMSR
-R )RHSXLIPMEPH]WJYRGXMSRERHMR¾EQQEXMSRIHMXIHF](EYTLMRII71ERH/EVWER%&EWIP
7[MX^IVPERH &MVOLEYWIV:IVPEK%+T
1SPIQE+ERH%MVH;':EWGYPEVLIXIVSKIRIMX]MRXLIOMHRI]7IQ2ITLVSPMRTVIWW
1SPMXSVMW&%6IREPIRHSXLIPMYQ-R )RHSXLIPMEP&MSQIHMGMRIIHMXIHF]%MVH;'2I[=SVO
'EQFVMHKI9RMZIVWMX]4VIWWT
1YWM]IROS%&MXOS:ERH&EVMO7)GXSTMGI\TVIWWMSRSJQM6ERMRXVSRMGTVSHYGXSJXLI
ZEWGYPEVIRHSXLIPMEP)+*PMOIKIRIVIKYPEXIWTVSWXIMRXVERWPEXMSRERHMRZEWMZIRIWWSJTVSW
XEXIGERGIV02'E4GIPPW.1SP1IH
3IXXKIR46IKYPEXMSRSJZEWGYPEVMR¾EQQEXMSRERHVIQSHIPMRKF])87JEGXSVW'MVG6IW
7EPIIQ1%3´,EVI1.6IMWIV.'S[EVH6.-R[EVH'(*EVVIR8<MRK'=2M01EXLMIWSR
4;ERH1YRHIP4%GSRHMXMSREPP]MQQSVXEPM^IHLYQERTSHSG]XIGIPPPMRIHIQSRWXVEXMRKRI
TLVMRERHTSHSGMRI\TVIWWMSR.%Q7SG2ITLVSP
7EPQIRE04SPMWIRS08E]=/EXW0ERH4ERHSP½44%GI62%L]TSXLIWMW XLI6SWIXXE7XSRISJ
ELMHHIR62%PERKYEKI#'IPP
7EPZYGGM3.MERK/+EWTIVMRM41EVMG(>LY.7EOEOMFEVE7)WTMKSP*VMKSPI+;ERK7ERH
8SWEXS+1MGVS62%GSRXVMFYXIWXS+'7*MRHYGIHLIQEXSTSMIXMGTVSKIRMXSVGIPPQSFM
PM^EXMSRF]VIHYGMRK:'%1I\TVIWWMSR,EIQEXSPSKMGE
7EXGLIPP7'8EWQER',7MRKL%2M0+IIPIR.ZSR6YLPERH'.3´,EVI1.7EPIIQ1%ZER
HIR,IYZIP04ERH1EXLMIWSR4;'SRHMXMSREPP]MQQSVXEPM^IHLYQERKPSQIVYPEVIRHSXLIPMEP
GIPPWI\TVIWWMRKJIRIWXVEXMSRWMRVIWTSRWIXS:)+*/MHRI]-RX
7GLEIJJIV:ERH%FVEWW'/1IGLERMWQWERHGSRXVSPSJTVSXIMRXVERWPEXMSRMRXLIOMHRI]%Q.
2ITLVSP
7YEVI^=ERH7IWWE;'1MGVS62%WEWRSZIPVIKYPEXSVWSJERKMSKIRIWMW'MVG6IW
7YEVI^=;ERK'1ERIW8(ERH4SFIV.7'YXXMRKIHKI 82*MRHYGIHQMGVS62%WVIKYPEXI
82*MRHYGIHI\TVIWWMSRSJ)WIPIGXMRERHMRXIVGIPPYPEVEHLIWMSRQSPIGYPISRLYQERIR
HSXLIPMEPGIPPW JIIHFEGOGSRXVSPSJMR¾EQQEXMSR.-QQYRSP
7YM;(EM=,YERK=0ER,=ER5ERH,YERK,1MGVSEVVE]EREP]WMWSJ1MGVS62%I\TVIWWMSR
MREGYXIVINIGXMSREJXIVVIREPXVERWTPERXEXMSR8VERWTP-QQYRSP
8SVVIW%+*EFERM11:MKSVMXS)ERH+EMX1.1MGVS62%JEXIYTSRXEVKIXMRK[MXLERXMQM62%
SPMKSRYGPISXMHIWEWVIZIEPIHF]ERMQTVSZIH2SVXLIVRFPSXFEWIHQIXLSHJSVQM62%HIXIG
XMSR62%
ZER1IYVW1;YPJIVX*1/RSP%.HI,EIW%,SY[IVXNIW1%EVXW04,.ERH1SPIQE+)EVP]
SVKERWTIGM½GIRHSXLIPMEPEGXMZEXMSRHYVMRKLIQSVVLEKMGWLSGOERHVIWYWGMXEXMSR7LSGO
ZER7SPMRKIR'7IKLIVW0&MNOIVO6(YMNW.16SIXIR1/ZER3IZIVIR6MIXHMNO%1&EIPHI
,.1SRKI1:SW.&HI&SIV,'5YE\4,6EFIPMRO8.ERHZER>SRRIZIPH%.%RXEKSQMV
1IHMEXIH7MPIRGMRKSJ)RHSXLIPMEP'IPP7TIGM½G1MGVS62%-QTEMVW-WGLIQME-RHYGIH%RK
MSKIRIWMW.'IPP1SP1IH
ZER7SPMRKIR'HI&SIV,'&MNOIVO61SRKI1ZER3IZIVIR6MIXHMNO%17IKLIVW0(I
:VMIW16ZERHIV:IIV)45YE\4,6EFIPMRO8.ERHZER>SRRIZIPH%.1MGVS62%QSHY
PEXIWIRHSXLIPMEP7(*I\TVIWWMSRERHQSFMPM^EXMSRSJ7GE 0MR TVSKIRMXSVGIPPWMRMW
GLEIQME'EVHMSZEWG6IW
:SW-,+SZIVW6+VSRI,./PIMN07GLYVMRO1HI;IKIV6%+SPHWGLQIHMRK6ERH6EFIPMRO
8.2*OETTE&HIGS]SPMKSHIS\]RYGPISXMHIWVIHYGIQSRSG]XIMR½PXVEXMSRMRVIREPEPPSKVEJXW
*%7)&.
;ERK+/[ER&'0EM*1'LSM4''LS[/10M4/ERH7^IXS''-RXVEVIREPI\TVIWWMSRSJ
QMGVS62%WMRTEXMIRXW[MXL-K%RITLVSTEXL]0EF-RZIWX
;ERK7%YVSVE%&.SLRWSR&%5M<1G%REPP].,MPP.%6MGLEVHWSR.%&EWWIP(YF]6ERH
3PWSR)28LIIRHSXLIPMEPWTIGM½GQMGVS62%QM6KSZIVRWZEWGYPEVMRXIKVMX]ERHERKMS
KIRIWMW(IZ'IPP
>EKIV6%.SLRWSR%'ERH0YRH79VIQMEMQTEGXWVIREPMR¾EQQEXSV]G]XSOMRIKIRII\TVIW
WMSRMRXLIWIXXMRKSJI\TIVMQIRXEPEGYXIOMHRI]MRNYV]%Q.4L]WMSP6IREP4L]WMSP *
*
>EQTIXEOM%/MIGLP7(VS^HSZ-;MPPIMX41E]V94VSOSTM11E]V%;IKIV73FIVLSPPIR^IV
*&SRSVE)7LEL%;MPPIMX.ERH1E]V14PEWQEQMGVS62%TVS½PMRKVIZIEPWPSWWSJIRHSXLIPMEP
QM6ERHSXLIVQMGVS62%WMRX]TIHMEFIXIW'MVG6IW
