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The long non-coding RNA MIAT regulates smooth muscle cell function and macrophage activity in
advanced atherosclerotic lesions
Yuhuang Li (Daniel), MD, PhDVascular Biology Unit
Department of Vascular and Endovascular SurgeryKlinikum rechts der Isar Technischen Universität München
Munich Vascular Conference, Dec 1-3, 2016
Background: Long non-coding RNA (LncRNA) and its function
<3%
>70% >200 nt
T. Thum, et al, Cir Res.2015~22 nt
Lars Maegdefessel et al.J Cell Physiol. 2011J Clin Invest 2012;Sci Transl Med 2012;Nat Commun 2014ATVB, 2015
miR-26amiR-21miR-24miR-29b
AtherosclerosisAneurysm
R.A.Boon, et al, JACC.2016
Role of lncRNA-MIAT (Myocardial Infarction Associated Transcript) in atherogenesis?
RNA profiling reveals highly increased lncRNA-MIAT in human carotid plaques
NA: normal arteryCP: carotid plaque
RNA profiling
Biobank of Karolinska Endarterectomies (BiKE)
NA
CP
Upregulated MIAT expression correlates with SMC content in human carotid plaque
NA
C
P
SMA
MIAT
SMA
MIAT
NA CP
NA: normal artery CP: carotid plaque
Rel
. MIA
T R
NA
exp
ress
ion
(F
old
ch
ange
)
Validated by q-PCR on additional n=77 BiKE patients
MIAT expression correlates with fate of SMCs in mouse plaque
4 w
Ligation
+ 4 d
Cuff
SMA SMA
MIAT
SMA SMA
NA
CP
NA
CP
MIAT
MIATMIAT
Ki67
Casp3
Ki67
Casp3
NA CP
Overexppressing lncRNA-MIAT promotes proliferation, inhibits apoptosis of HctSMC
40
50
60
70
80
90
0 8 16 24 32 40 48 56 64 72
NC MIAT
0
2
4
6
8
10
0 8 16 24 32 40 48 56 64 72
NC MIAT
Rel
. MIA
T ex
pre
ssio
n
NC
MIATP
rolif
era
tion
(Confluen
ce %
)
hr
Ca
sp
3
A CA
pop
tosis
(Casp3+
/ m
m2)
C
hr
D
B
0
1
2
NC MIAT
900
1100
Knocking down lncRNA-MIAT suppresses proliferation, induces apoptosis of HctSMC
30
50
70
90
110
0 8 16 24 32 40 49 57 65
SCR MIAT KD
Rel
. MIA
T ex
pre
ssio
n
0
0,2
0,4
0,6
0,8
1
1,2
Pro
lifera
tion
(Confluen
ce %
)
hr
0
20
40
60
0 8 16 24 32 40 48 56 64 72
SCR MIAT KD
Apo
pto
sis
(Casp3+
/ m
m2)
C
hr
A CB
Michalik, et al, Circ Res. 2014
NC
MIAT KDCa
sp
3
D
A Transcriptional factor array (free energy <-30 kcal/mol )
lncRNA-MIAT regulates HctSMC functions via regulating transcription factor?
B MIAT effects upon modulation of TF→Abolish single effect on cell fates (IncuCyte)→EGR1 siRNA/GapmeRs?
LncRNA-MIAT
VSMCs
Proliferation
Apoptosis
Carotid plaque development
Via EGR1?
Rel
. mR
NA
exp
ress
ion
(Fo
ld)
MIAT expression correlates with Macrophages in human advanced plaques
Stable Rupture
MIATMIAT
CD68CD68
MIATMIAT
CD68CD68
MRI BiobankDr. Jaroslav Pelisek
MIAT expression correlates with Macrophages in mouse advanced plaques
F4/80
MIAT
F4/80
MIAT
Stable Rupture
Knocking down lncRNA-MIAT impaires oxLDL uptakeof human Macrophage
Rel. M
IAT
expre
ssio
n
oxL
DL
up
take
hr
NC MIAT KD
0
200
400
600
800
1000
1200
0 4 8 12 16 20 24
NC MIAT KD
0
0,2
0,4
0,6
0,8
1
1,2
NC MIAT KD
FIT
C-o
xLD
L
Knocking down lncRNA-MIAT impaires oxLDL uptakeof mouse Macrophage
0
50
100
150
200
250
0 5 10 15 20
NC MIAT KDo
xL
DL
up
take
hr
NC MIAT KD
Rel. M
IAT
expre
ssio
n
0
0,2
0,4
0,6
0,8
1
1,2
NC MIAT KD
FIT
C-o
xLD
L
A
B1. IF staining/WB: NF-KB activation upon MIAT knockdown with/without oxLDL2.MIAT effects upon pathway specific inhibitor
C
QPCR: MIAT level in subtype of microphages (PMA-THP1)
Pro-inflammatory properties of lncRNA-MIAT via regulating Macrophage
LncRNA-MIAT
Macrophage
oxLDL uptake
Inflammatory potent
Carotid plaque progression & unstability
Cytokines array
0
2
4
NC LPS
0
0,5
1
1,5
NC IL4
Rel. M
IAT
expre
ssio
n
M1 M2
A
B
lncRNA-MIAT regulates SMC → Macrophage?
C Effects of MIAT after modulation of KLF4Expression: SM22a, MYH11, ACTA2; Mac2, CD68; Cytokines; Collagens
Rel
. mR
NA
exp
ress
ion
(Fo
ld)
KLF4
pELK-1
G-C Repressor
SM22α Promoter
HDAC2
Phenotypic Switching
KLF
4
NC MIAT MIAT KDSCR
Circ Res. 2012Nat Med. 2015
Summary:LncRNA-MIAT promotes atherosclerotic lesions via regulating
smooth muscle cell functions and macrophage activity
VSMCs
Proliferation
Apoptosis
Carotid plaque development
LncRNA-MIAT
Macrophage
oxLDL uptake
Inflammatory potent
Carotid plaque progression & unstability
Phenotypic Switching
Vascular Surgery, KIUlf HedinClaes BergmarkLjubica PerisicSilvia Aldi
Molecular Vascular Medicine, KIKatja ChernogubovaHong JinAlexandra BäcklundSuzanne M. EkenAlbert BuschChangyan SunNancy SimonAnna OlofssonGreg KorzunowiczLars Maegdefessel
Philip S. TsaoRonald L. DalmanJoshua M. SpinNicholas J. Leeper
Atherosclerosis Research Unit, KIPer ErikssonPeter GustafssonOlivera WerngrenEwa EhrenborgXintong JiangGabrielle Paulsson- BerneGöran K. Hansson
Vascular Biology Unit, TUMLars MaegdefesselJaroslav PelisekShengliang LiuRenate Hegenloh
Genomic structure and evolutionary conservation
Rapicavoli et al. BMC Developmental Biology 2010, 10:49
Treatment of NFKB inhibitor impaires oxLDL uptakeof mouse Macrophage
oxL
DL
up
take
hr
NFKBi-
oxLDL
Macrophage
0
1
2
3
4
5
0 2 4 6 9 11 13 15
NFKBi-
NFKBi+
NFKBi+
LncRNADisease database
lncRNA-myocardial infarction-associated transcript (MIAT)
lncRNA-MIAT regulates microvascular dysfunction by functioning as a competing endogenous RNA (miR-150, VEGF) (Circ Res. 2015;116:1143-1156.)
Mild but significantly positive correlations of MIAT with markers of T-lymphocytes, inflammation, apoptosis and matrix degradation in carotid plaques. All correlations with other typical markers of cells and pathways were ns.
Correlations with markers in BiKE
CASP9 IL17FTGFB1BCL2
MMP2
MMP24
MMP25
FOXP3
ADAMTS