Cell Reports, Volume 7

Supplemental Information

Dysregulation of the MiR-324-5p-CUEDC2 Axis

Leads to Macrophage Dysfunction

and Is Associated with Colon Cancer

Yuan Chen, Shao-Xin Wang, Rui Mu, Xue Luo, Zhao-Shan Liu, Bing Liang, Hai-

Long Zhuo, Xiao-Peng Hao, Qiong Wang, Di-Feng Fang, Zhao-Fang Bai, Qian-Yi

Wang, He-Mei Wang, Bao-Feng Jin, Wei-Li Gong, Tao Zhou, Xue-Min Zhang,

Qing Xia, and Tao Li

Figure S1

A

Cuedc2 +/+ +/- -/-

13.5KB

10.7KB

B

C

LoxP

mCUEDC2

b-actin45KD

35KD

D

10 0 10 1 10 2 10 3 10 4

FITC

Key Name Parameter Gat

DATA.005 FL1-H G1

DATA.006 FL1-H G1

10 0 10 1 10 2 10 3 10 4

FITC

Key Name Parameter Gat

DATA.005 FL1-H G1

DATA.006 FL1-H G1

Peritoneal -Mf

F4/8090.7%

100 102 103 104101

Counts

04

08

01

20

16

02

00

IgGF4/80

E

M0 M2M1

Arginase I

a-tubulin55KD

35KD

130KD iNOS

M0 M2M1

WT Cuedc2-/-

WT

KO

Exon 2 345 6 78 9

EcoRVEcoRV

WT: 13.5 KB

5’ 3’

Cuedc2-/-: 10.74 KB

EcoRVEcoRV

5’ 3’

WT

Monocyte BMDM

Cuedc2-/-

Ly6C

82.3%

Co

un

ts0

30

60

12

01

50

100 102 103 104101

90

104

F4/80

12.7%

100 102 103101

Co

un

ts

104

Ly6C

22.5%

100 102 103101

Co

un

ts

F4/80

83.7%

100 102 103 104101

Co

un

ts2

04

06

08

00

81.2%

100 102 103104101

Co

un

ts1

02

03

05

04

00

21.7%

100 102 103 104101

Co

un

ts0

104

10.3%

100 102 103101

Co

un

ts

86.1%

100 102 103 104101

Co

un

ts0

30

60

12

01

50

90

03

06

01

20

15

09

01

00

40

60

80

20

40

80

12

02

00

16

00

80

16

02

40

40

03

20

0

100KD

35KD

70KD

0 5 10 15 30 600 10LPS (min)

p-IKKa/b

mCUEDC2

a-tubulin

WT

5 15 30 60

IKKa

Cuedc2-/-

a-tubulin

55KD

100KD

70KD

55KD

0

20

40

60

80

100

0

10000

20000

30000

0

500

1000

1500

2000

IL-6

mR

NA

(re

lative)

IL-1

2p40 m

RN

A (

rela

tive)

IL-1b

mR

NA

(re

lative)

M0

IL-6 TNFa IL-12p40

0

10

20

30

40

0

1000000

2000000

3000000

4000000

0

10

20

30

40

CC

L17 m

RN

A (

rela

tive)

FIZ

Z1 m

RN

A (

rela

tive)

YM

-1 m

RN

A (

rela

tive)

CCL17 FIZZ1 YM-1

F

G

WT

Cuedc2-/-

Monocyte BMDM

Ly6C F4/80 Ly6C F4/80

WT

Cuedc2-/-

M1 M0 M1 M0 M1 M0 M2 M0 M2 M0 M2

Figure S1. CUEDC2 Controls the Production of Pro-inflammatory Cytokines

through Regulating NF-B Signaling, Related to Figure 1

(A) Cuedc2 gene scheme of WT and knockout (KO) alleles, locations of EcoR V

digesting sites and probe are indicated. Targeting vector was designed to delete the

LoxP-flanked sequences to remove exons 2–9.

(B) Southern blot analysis of EcoR V digested genomic DNA from mice, a single

band at 13.5 kb indicating WT (+/+), a single 10.7 kb band indicating homozygous

(-/-), and both bands, heterozygous (+/-).

(C) Primary monocytes were obtained from WT and Cuedc2-/-

mice and were

differentiated to BMDMs. Cells were stained with Ly6C, the monocyte marker, and

F4/80, the macrophage marker before and after differentiation, respectively, and the

differentiation efficiency was verified by flow cytometry analysis.

(D) Immunoblot analysis of CUEDC2 in monocytes, BMDMs and peritoneal

macrophages (p-Mf), b-actin blot indicates the loading of lanes. Flow cytometry

analysis of F4/80 staining of p-Mf to verify the cell purity.

(E) Immunoblot analysis of iNOS and Arginase I in BMDMs from WT and Cuedc2-/-

mice, M0（untreated macrophage）M1 (polarized with LPS 100 ng/ml and IFN 20

ng/ml), M2 (polarized with IL-4 20 ng/ml).

(F) BMDMs from WT and Cuedc2-/-

mice were polarized to M1 and M2, and the

associated genes for each type were detected by qPCR. Error bars, SEM.

(G) BMDMs from WT and Cuedc2-/-

mice were treated with 10 ng/ml LPS for

indicated times. Immunoblot analysis of phosphorylated IKKa/bwhich indicating

the activation of NF-B signaling. Loading of lanes was indicated by a-tubulin blots.

Separated immunoblot analysis of IKKa with the same samples was shown as

additional loading for phosphorylated IKKa/b

Figure S2

C

B

Human 5’-GGCUCAGAUCCCAGAGGGAUGCA-3’

Chimpanzee 5’-GGCUCAGAUCCCAGAGGGAUGCA-3’

Monkey 5’-GGCUCAGAUCCCAGAGGGAUGCA-3’

Mouse 5’-ACTGCUGAUCCCAGAGGGAUGCA-3’

miR-324-5p 3’-UGUGGUUACGGGAUCCCCUACGC-5’

CU

ED

C2

3’

UT

R

BMDM

mCUEDC2

a-tubulin

Ctrl 324-5p

Mimic

55KD

35KD

A

CUEDC2 3’ UTR

miR-324-5p

miR-330-5p

miR-326

3’ poly(A)Stop codon

D

E F

0

0.4

0.8

1.2

hC

UE

DC

2 m

RN

A (

rela

tive

)

Macrophage

hCUEDC2

324-5pCtrl

0

0.5

1

1.5

2

2.5

324-5pCtrl

De

nsito

me

try

of

CU

ED

C2

/a-tu

bu

lin

p = 6.091E-03

Inhibitor:

Inhibitor

Ctrl 324-5p Ctrl 324-5p 324-5pCtrl

55KD

35KD

1.00 2.22

hCUEDC2

a-tubulin

1.86 2.161.00 1.00

# 1 # 2 # 3

Monocyte

Monocyte

G

CUEDC2

a-tubulin55KD

35KD

Flag-CUEDC2

Macrophage

Figure S2. MiR-324-5p Regulates the Differential Expression of CUEDC2

between Monocytes and Macrophages, Related to Figure 3

(A) Distribution of predicted target sites for three relevant microRNAs in 3’UTR of

CUEDC2 mRNA as indicated.

(B) Immunoblot analysis of lysates of monocytes 24 hours after transfection of

miR-324-5p inhibitor or control inhibitor (Ctrl), numbers below lanes are

densitometry (hCUEDC2 relative to a-tubulin).

(C) Densitometric analysis of band intensity of CUEDC2 band relative to a-tubulin,

error bars, SEM.

(D) MiR-324-5p-binding sites in 3’UTR of CUEDC2 mRNAs is conserved in

mammals.

(E) Immunoblot analysis of CUEDC2 in miR-324-5p mimic or control (Ctrl) mimic

transfected mouse BMDMs, a-tubulin blots indicting loading of lanes.

(F) Human macrophages (Mf) were transfected with miR-324-5p mimic or control

(Ctrl) mimic, abundance of CUEDC2 mRNA was analyzed with qPCR. Data are

presented as relative quantification compared to GAPDH. Data represent three

independent experiments, error bars, SEM.

(G) Human macrophages were transfected with control or miR-324-5p mimics for 24

hours, and then the cells were infected with lenti-virus expressing Flag-tagged

CUEDC2 or Flag vector. Cell lysates were then collected for immunoblot analysis of

CUEDC2, a-tubulin blots indicting loading of lanes.

Figure S3

A

WT Cuedc2-/- WT Cuedc2-/-

-DSS +DSS

B

WT

Cuedc2-/-IL-1b

IL-1b

mR

NA

(re

lative

)

Colon

*

IL-6

IL-6

mR

NA

(re

lative

)

Colon

**

TNFa

TN

Fa

mR

NA

(re

lative

) *

Colon

0

0.5

1

1.5

2

2.5

3

Time (days)

1 2 3 4 5 6 7 8 9

**

**

Ble

ed

ing

Sco

re

WT

Cuedc2-/-

0

0.5

1

1.5

2

2.5

3

Time (days)

Sto

ol S

co

re

1 2 3 4 5 6 7 8 9

****WT

Cuedc2-/-

D

C

E

Mf transplantation

DSS

Sacrifice

D-1 D0 D1 D6 D10

Mf transplantation

0

2

4

6

8

0

1

2

3

0

5

10

15

Figure S3. CUEDC2 Expression in Macrophages is Critical for Protection

against Colitis, Related to Figure 4

WT and Cuedc2-/-

mice were subjected to oral administration of 2.5% DSS for 6 days,

followed by regular drinking water for 3 days.

(A-B) Rectal bleedingscore (A) and stool consistency scores (B) were scored daily,

Cuedc2-/-

mice (n = 11) and WT mice (n = 12), data represent means ± SEM.

(C) Colon length of WT and Cuedc2-/-

mice, untreated (left panel) and DSS treated

(right panel).

(D) 9 days after DSS administration, colon tissues from WT and Cuedc2-/-

mice were

obtained and subjected to qPCR analysis for mRNA levels of IL-6, TNFa and IL-1b.

Date represent three independent experiments, error bars, SEM.

(E) Schematic overview of macrophage transplantation regimen.

Figure S4

Figure S4. CUEDC2 Expression in Colon Cancer Cells and Adjacent Normal

Epithelial Cells, Related to Figure 5

(A) Representative images from immunohistochemical staining of CUEDC2 in

adjacent normal tissues and tumor tissue of CRC. The boxed areas (“1” and “2”) in

the left image are magnified as the right images to show the adjacent normal tissue

and carcinoma areas. Scale bars = 100 m.

(B) CUEDC2 expression scores are shown as box plots. The horizontal lines in the

box representing the median, the boxes representing the first and third quartile,

respectively, and the vertical bars representing the range of data. CUEDC2 expression

in tumor was compared to that in adjacent normal tissues with Mann-Whitney U test,

n = 36.

BA

hC

UE

DC

2 S

co

re IEC

0.5

1.0

1.5

2.0

2.5

3.0

p = 0.5460

0

CarcinomaAdjacent normal

hCUEDC2

Colorectal Cancer

1

1 2

2

CarcinomaAdjacent normal

Figure S5

Figure S5 IL-4 regulates miR-324-5p expression through c-MYC, Related to

Figure 6

(A) Human macrophages were treated with IL-4 (20 ng/ml) for 12 hours, and cell

lysates were subjected to immunoblot analysis for c-MYC protein level.

(B) C-MYC or non-targeting (NT) siRNAs transfected macrophages were treated with

IL-4 for 24 hours, followed by qPCR analysis of miR-324-5p. Error bars, SEM.

(C) Immunoblot analysis of c-MYC in siRNAs transfected macrophages in (B),

b-actin blots indicate loading of lanes (A and C).

A

miR

-32

4-5

p (

rela

tive

)

IL-4-

B

IL-4-

c-MYC

b-actin40KD

55KD

siRNA:

c-MYC

b-actin40KD

55KD

c-MYCNT

C

0

0.5

1

1.5

2

2.5siRNA-NT

siRNA-c-MYC

Figure S6

Figure S6. Increased Colitis-Associated Colorectal Tumorigenesis in CUEDC2

Deficient Mice, Related to Figure 7

(A) Average number of total tumor number per mouse was determined at day 84. Data

represent means SEM, n = 22.

(B) Overall grading of hyperplasia of WT and Cuedc2-/-

mice.

A

0

20

40

60

80

100

120

servere

moderate

low

% o

f m

ouse w

ith

hyp

erp

lasia

WT Cuedc2-/-

B

p = 3.5733E-7

WT Cuedc2-/-0

4

8

12

16

Tu

mo

r n

um

be

r

Supplemental Experimental Procedures

Reagents

FITC-anti-F4/80 (BM8), PE-anti-Ly6C (HK1.4), PE-anti-B220

(RA3-6B2) and FITC-anti-CD3 (17A) were from Biolegend.

PE-anti-Ly6G (RB6-8C5) was from eBioscience. Anti-mouse iNOS

(610430), rmIFN(554587) were from BD Biosciences.

Anti-mouse-Arginase I (sc-20150) and anti-b-actin (C-11) were from

Santa Cruz. Anti-pIKKa/b (2681), anti-IKKa (2682) and anti-a-tubulin

(2144) were from Cell Signaling Technology. Anti-c-MYC (A00172-200)

was from Genscript. RmIL-4 (404-ML) and rhIFN(215-IF) were from

R&D systems.

Generation of Cuedc2-/- Mice

The targeting construct was designed to flank Cuedc2 exon 2-9 (2.76 kb

fragment) with loxP sites. The long homology arm of 5.14 kb DNA

fragment 5’ to exon 2 of Cuedc2 and the short homology arm of 2.05 kb

fragment 3’ to exon 9 were sub-cloned into a targeting vector. A loxP site

was inserted 5’ to exon 2 and a loxP/FRT-flanked neomycin (neo)

resistance cassette was inserted 3’ to exon 9. 10 g of the targeting

constructs were linearized by NotI and then transfected by electroporation

to BA1 (C57BL/6 x 129/SvEv) hybrid embryonic stem cells. After

selection with G418 antibiotics, surviving clones were expanded for PCR

and Southern blot analysis to identify recombinant ES clones. Positive

clones were microinjected into C57BL/6 blastocysts and transferred into

CD-1 foster mothers. The resulting male chimeras were mated with

wild-type C57BL/6 females to test for germline transmission.

F1 agouti mice were genotyped by PCR, and the mice with

Cuedc2 fl-neo/+

genotype were crossed with FLP-deleter transgenic mice

(stock number 003946, Jackson Laboratory) to remove the Neo cassette

by the FLP-mediated recombination. The single floxed Cuedc2 allele was

converted to a null allele by Cre-mediated recombination (EIIa-Cre, stock

number 003724，Jackson Laboratory). Genotypes were analyzed by PCR

and confirmed by Southern blot analysis of EcoR V-digested tail genomic

DNA (10 μg), yielding 13.5- and 10.7-kb fragments for the Cuedc2+/+

and

Cuedc2-/-

alleles, respectively.

Flow Cytometry

Cells were washed in ice-cold flow-cytometry buffer (0.5% BSA and 2

mM EDTA in PBS, pH 7.2), and incubated with each antibody for 30

minutes followed by washing with flow-cytometry buffer. Data were

acquired on a FACSArial II flow cytometer and analyzed with CellQuest

Pro (BD Biosciences).