Constitutive Expression of microRNA -150 in Mammary ...Constitutive Expression of microRNA -150 in...

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)


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


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


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


Lipid Synthesis Transcripts are Reduced by Constitutive miR-150 Expression


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


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


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


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


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

Constitutive Expression of microRNA -150 in Mammary ...Constitutive Expression of microRNA -150 in...

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