View
3
Download
0
Category
Preview:
Citation preview
Constitutive Expression of microRNA-150 in Mammary Epithelium Suppresses
Secretory Activation and Impairs De NovoLipogenesis
byRichard Heinz, Ph.D.
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
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
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)
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
Predicted Targets of miR-150 are Involved in Lipid and Cholesterol Biosynthesis Pathways
Heinz, Development (2016)
HypothesisThe decrease in miR-150 is necessary to allow for the
translation of transcripts critical for lactation.
Pregnancy Lactation
miR
-150
Lev
els
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.
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)
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)
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
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)
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)
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)
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)
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)
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.
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
Recommended