Constitutive Expression of microRNA -150 in Mammary ...Constitutive Expression of microRNA -150 in...
Transcript of Constitutive Expression of microRNA -150 in Mammary ...Constitutive Expression of microRNA -150 in...
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Constitutive Expression of microRNA-150 in Mammary Epithelium Suppresses
Secretory Activation and Impairs De NovoLipogenesis
byRichard Heinz, Ph.D.
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Principles of miRNA Biology
Adapted from Peter, Oncogene (2010)
miRNA
mRNA a mRNA b mRNA c
miRNA 1 miRNA 2 miRNA 3
mRNA
mRNA Target Cleavage
Translational Repression
mRNA Deadenylation
miRNA Manifest as decreasedprotein product
of the targeted gene
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Adapted from Avril-Sassen et al., BMC Genomics (2009)and Rudolph et al., J Mammary Gland Biol Neoplasia (2003)
PregnancyDay 14
LactationDay 2
Mam
mar
y G
land
R
NA
Leve
ls
miRNAs
LactationmRNAs
Decrease in miRNAs at Late PregnancyCoincides With Increased Expression
of mRNAs Critical for Lactation
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Legend - HeatmapColor Range
Low 0 High
miR-150-5p
miR-532-5p
miR-146b-5pmiR-181b-5pmiR-706miR-494-3pmiR-143-3pmiR-31-5pmiR-181a-5p
miR-342-3pmiR-20b-5pmiR-146a-5pmiR-361-5pmiR-342-5pmiR-140-3pmiR-191-5pmiR-425-5pmiR-155-5pmiR-18a-5pmiR-106a-5pmiR-17-3pmiR-130b-3p
miR-652-3pmiR-150-3pmiR-29a-3pmiR-20a-5pmiR-106b-5pmiR-15b-5pmiR-185-5pmiR-106b-3p
miR-92a-3pmiR-17-5p
LactationDay 2
PregnancyDay 14
Many miRNAs Decrease by 2 Fold or MoreBetween Pregnancy and Lactation
Heinz, Development (2016)
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Legend - HeatmapColor Range
Low 0 High
miR-150-5p
miR-532-5p
miR-146b-5pmiR-181b-5pmiR-706miR-494-3pmiR-143-3pmiR-31-5pmiR-181a-5p
miR-342-3pmiR-20b-5pmiR-146a-5pmiR-361-5pmiR-342-5pmiR-140-3pmiR-191-5pmiR-425-5pmiR-155-5pmiR-18a-5pmiR-106a-5pmiR-17-3pmiR-130b-3p
miR-652-3pmiR-150-3pmiR-29a-3pmiR-20a-5pmiR-106b-5pmiR-15b-5pmiR-185-5pmiR-106b-3p
miR-92a-3pmiR-17-5p
LactationDay 2
PregnancyDay 14
Decrease in miR-150 Coincides WithIncrease of miR-150 Predicted Targets
Lao1Slc39a8
OlahGldcQsox1Fam13aScd2Kcnk6Odc1
Fasn
Scd1
LbpFads1
Btn1a1Elovl5
Car5b
Cxcl14Irs1
Mfge8
Pcx
Esyt3
Tcn2Slc25a1
Egf
Prkaa2
Aldoc
Tspan7
Fam160a2
LactationDay 2
PregnancyDay 14
PredictedTargets
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Predicted Targets of miR-150 are Involved in Lipid and Cholesterol Biosynthesis Pathways
Heinz, Development (2016)
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HypothesisThe decrease in miR-150 is necessary to allow for the
translation of transcripts critical for lactation.
Pregnancy Lactation
miR
-150
Lev
els
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Pregnancy Lactation
miR
-150
Lev
els
HypothesisThe decrease in miR-150 is necessary to allow for the
translation of transcripts critical for lactation.
Constitutive expression of miR-150 is predicted to override the natural decline and result in defective
lactation, which would lead to decreased weight gain or survival of offspring.
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Transgenic Mouse Model to Constitutively Express miR-150 in the
Mammary Gland During Lactation
WAP-Cremammary epithelial cell specific expression of Cre recombinasefrom late pregnancy through lactation
Stop-150
Heinz, Development (2016)
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3 5 7 9 1 1 1 3 1 50
5 0
1 0 0
D a y s o f L a c ta t io n
% S
urv
iva
lB
iolo
gic
al P
up
s
n = 5 l it te rs
n = 8 lit te rs
p < 0 .0 0 0 1
Survival of Fostered PupsSurvival of Biological Pups
Constitutive miR-150 ExpressionDramatically Reduces Pup Survival
Controls
ConstitutivemiR-150 Expressing
Dam Genotype
3 5 7 9 1 1 1 3 1 50
5 0
1 0 0
D a y s o f L a c ta t io n%
Su
rviv
al
Fo
ste
r P
up
s
n = 5 l it te rs
n = 5 lit te rs
p < 0 .0 0 0 1
Heinz, Development (2016)
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Lipid Synthesis Transcripts are Reduced by Constitutive miR-150 Expression
Legend - HeatmapColor Range
Low 0 High
Insig1Fasn
Scd2Acly
Elovl6AcacbPank3Fads2
LplFads1AcacaAldocElovl5Thrsp
Elovl1Agpat1
Me1Scd1
Slc2a1
Fabp3Olah
Slc25a1
PregnancyDay 14
LactationDay 2
Normal MiceConstitutive miR-150
ExpressingControls
Transgenic Mice at Lactation Day 2
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C o n tro l C o n s t itu t ivem iR -1 5 0 E xp re s s in g
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5p = 0 .0 0 0 1
FA
SN
No
rma
lize
d t
oα
-Tu
bu
lin
FASN
Control
α-Tubulin
Quantification of FASN at L2
Constitutive miR-150 Expression SuppressesFatty Acid Synthase (FASN) at Lactation Day 2
FASN synthesizes all de novo fatty acids.Constitutive
miR-150 Expressing
Heinz, Development (2016)
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ControlFASN IHC
Constitutive miR-150 Expressing
P14
P14
L2
L2
Constitutive miR-150 Expression SuppressesFatty Acid Synthase (FASN) at Lactation Day 2
Scale Bar = 50μm400× magnification
Heinz, Development (2016)
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miR-150-5p Targets the Predicted Sitein Fasn 3′UTR
Luciferase Reporter Assay
W T m u t0 .0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
Re
lativ
e L
uci
fera
se U
nits
p = 0 .0 0 8
N Cm iR -1 5 0
p = 0 .2
Putative miR-150 binding site on mouse Fasn 3′UTR
378: 5’-…cagaacccccuaaaaugggagaa…-3’
3’-gugaccauguucccaacccucu-5’
miR-150-5p
*** *mut:cta c
Heinz, Development (2016)
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miR-150-5p Targets the Predicted Sitein Fasn 3′UTR
Luciferase Reporter Assay
W T m u t0 .0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
Re
lativ
e L
uci
fera
se U
nits
p = 0 .0 0 8
N Cm iR -1 5 0
p = 0 .2
Putative miR-150 binding site on mouse Fasn 3′UTR
378: 5’-…cagaacccccuaaaaugggagaa…-3’
3’-gugaccauguucccaacccucu-5’
miR-150-5p
*** *mut:cta c
Heinz, Development (2016)
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Constitutive miR-150 Expression SuppressesDe Novo Fatty Acid Synthesis
1 8 :1 n
-9
1 8 :2 n
-6
1 8 :3 n
-3
1 8 :3 n
-60
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
D ie ta ry F a tty A c id s
A c y l C h a in L e n g th a n d S a tu ra t io n
mo
l/mo
l TA
G1 0 :0
1 2 :01 4 :0
1 6 :01 8 :0
0
2 0 0
4 0 0
6 0 0
S a tu ra te d F a tty A c id s
A c y l C h a in L e n g th
mo
l/mo
l TA
G
C o n tro l
C o n s titu tivem iR -1 5 0 E x p re s s in g
*** *
*
**
Isolated L2 MECs Lipid Extraction Gas ChromotographyMass Spectrometry
Heinz, Development (2016)
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Conclusions
Forced expression of exogenous miR-150 in mouse mammary glands during late pregnancy, to override the natural decline in this miRNA at lactation….
• causes decreased pup survival.
• suppresses FASN, ACACA, and OLAH protein.
• suppresses de novo lipid synthesis.
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Neville LabPeggy Neville
Palani RamanathanPatty Webb
FundingNIH NICHD P01
PAR-10-245 Functional Development of the
Mammary Gland
NIH/NCATS Colorado CTSI Grant Number TL1
TR001081
AcknowledgementsRicher Lab
Jennifer RicherMichael GordonNicole SpoelstraBeatrice BabbsKiel Butterfield
Michael Rudolph
Gu LabHaihua Gu
Hongwei GaoShang Chen