The Center for Biomedical Research

Research Experimental Design

Cong-Yi Wang ( ), MD, PhD
Professor
Director, the Center for Biomedical Research
Director, Tongji Biobank
Tongji Hospital, Tongji Medical College
Huazhong University of Science & Technology
Wuhan, China
Email: wangcy@tjh.tjmu.edu.cn

The Center for Biomedical Research

(Faculty & Staff)

(Graduate Students)

2014

2014

2014

2015

2015
Mohammed

2014

2015

2015

2015

2015

2015

2015

2016

2014

2016

2015

2016

2016

2016

Research Directions
Using the advanced high throughput genomic and proteomic approaches
to dissect the biological questions underlying disease etiologies and to
develop effective therapeutic strategies aimed at disease
prevention/intervention.
(I) Diabetes and Diabetic Complications
1. Beta cell Biology
2. Genetic and epigenetic factors in disease pathoetiology.
(II) Autoimmune Diseases and Allograft Rejection
1. Innate immunity in allograft rejection
2. Innate immunity in autoimmune initiation and progression
3. Epigenetic factors and PTM in the regulation of auto/alloimmune
response.
(III) Genomic and Proteomic Studies
1.
High throughput whole genome RNA/DNA sequencing
1. Comparative proteomic analysis of PTM.

Experimental Design

Science answers questions with experiments

Clinical Case

Parents: Nomal
3-year daughter: Normal
Maffuccis syndrome

Ollier disease

TTACTTGATCCCCATAAGCATGACGACCTATGATGATAGGTTTTACCCATCCACT

A
Normal

Protein muation: R132->C mutation

Tumor tissue : 19:12 (Reference:mutant)
Blood Sample : 37: 0 (Reference:mutant)
Mutation Ratio: 38.7%

Mu
IDH1 mutation G->A

??????

TTTAGGCGGCTCCAAGCCGAGCATGACAGGCAGGCT

Normal

Mu

EXXX2 mutation A->T

Protein muation: L309->I mutation

Tumor tissue : 61:19
Blood Sample : 66: 0
Mutation Ratio: 23.8%

Experimental Design

1. Reference checking (Accumulation of your

knowledge in the field);
2. Pilot experiments to obtain a specific phenotype
(positive control and negative control);
3. Experiments for dissecting the underlying
mechanism.

Define a Problem to Solve

Begin by asking a question

about your topic
What is a good question for an
experiment?

One that is testable with the

materials at hand

Create a hypothesis to guide

your investigation.

What is a hypothesis?

Your best thinking about how the change

you make might affect another factor.
Tentative or trial solution to the question.
An if then statement.

Scientific Experimental Design

1. Reference checking;
2. Perform experiments to obtain a specific phenotype
(positive control and negative control);
3. Experiments for dissecting the underlying
mechanism.

Nuclear Genome

What are we
missing behind
the genomic
sequence?

Epigenome

Vascular diseases:

unknown

MethylCpG Binding Domain (MBD)

Proteins
Methyl-CpG Binding Domain
(MBD) Proteins: MeCP2, MBD1,
MBD2, MBD3, MBD4
MeCP2-/-: Rett syndrome
symptoms (a neurodegenerative
disease); neural differentiation
MBD1-/-: almost normal
phenotype, decreased neuronal
differentiation & chromosome
instability; neural differentiation
MBD2-/-: almost normal
phenotype in the physiological
condition
MBD3-/-: embryonic lethal
MBD4-/-: more C to T transitions,
enhanced tumorigenesis; DNA repair

Methyl-CpG Binding Domain Protein 2 (MBD2)

Hendrich et al. Genes Dev. 2001 March 15; 15(6): 710723.

Scientific facts:
1.MBD2 itself does not affect the
methylation of DNA;
2.It serves as an interpreter to
decipher the information encoded
by the DNA methylome;
3.animals deficient in MBD2 failed to
show perceptible phenotype in the
physiological condition.
Hypothesis:
MBD2 is an ideal target to dissect
the role of DNA methylation in the
regulation of endothelial function in

An siRNA dose-dependently
suppressed MBD2 expression
in HUVECs

-actinMBD2

siRNA:

Ctl

MBD2

100nM

50nM

Rao et al. Circulation, 2012

100nM

**

Knockdown of MBD2 enhances endothelial a

Human Umbilical Vein Endothelial cells: HUVECs

MBD2 siRNA

Control
siRNA

**

siRNA:

Tube Formation

Rao et al. Circulation, 2012

Ctl

MBD2

siRNA:

Ctl

Proliferation

MBD2

Knockdown of MBD2 expression prevents

ECs against H2O2-induced apoptosis

P
I

120409 apoptosis.002

Mbd2 siRNA

Control
siRNA

15.1

6.22

1204091 apoptosis.001
100
10
102
103
104
ANNEXIN 4.65

5.81
100

Rao et al. Circulation, 2012

101

102
103
ANNEXIN

10
Annex-V

siRNA:

Ctl

MBD2

Suggestive Conclusion
MBD2 plays a predominant role in
deciphering DNA methylome-encoded
information in endothelial cells;
DNA methylation changes are present
in endothelial cells which play a role
angiogenesis;
Knockdown of MBD2 releases the
repressive effect of DNA methylation on
those angiogenic genes, leading to
enhanced angiogenesis.

Once you got suggestive phenotype, more

studies are needed to confirm the phenotype

In vivo studies

How to dissect the implication of

DNA methylation in endothelial
dysfunction in disease sate?
Two models:
Hindlimb ischemic injury
Oxidative Stress

Diabetic endothelial dysfunction

Ischemic insult time-dependently

upregulates MBD2 expression
Days after hindlimb ischemic injury
7

10

2
14

GAPDH

MBD2

Xiaoquan Rao

Jixin Zhong
Rao et al. Circulation, 2012

MBD2 -/-

WT

Loss of MBD2 Protects Mice against Hindlimb Is

Before
day7
surgery

after
day14
surgery

Rao et al. Circulation, 2012

day2

day4

0
1000

Mice deficient in MBD2 show enhanced

capillary and arteriole formation after
ischemic surgery

CD31

Mbd2-/-

WT

Mbd2-/-

arterioles/field

-Smooth muscle actin

WT

WT

Rao et al. Circulation, 2012

Mbd2-/-

WT

Mbd2-/-

Mice Deficient in MBD2 Are Completely Protected from Diabetesinduced Endothelial Dysfunction
A

KCL (120mmol/L

Contractile response

Exogenous NO (SNP)
rescued endothelial
relaxation

Endothelial dependent relaxation

Summary
1. Ischemic injury induces MBD2 expression
in endothelial cells along with enhanced
capillary and arteriole formation;
2. Loss of MBD2 provides protection against
hindlimb ischemic injury;
3. Mice deficient in MBD2 are completely
protected from diabetes-induced
endothelial dysfunction.

Experimental Design

1. Reference checking;
2. Perform experiments to obtain a specific phenotype
(positive control and negative control);
3. Experiments for dissecting the underlying
mechanism (generate a story).

Key question:

What are the underlying mechanism

Proangiogenic signals
Survival signals

Suppression of MBD2 Activates EC

Survival and Proangiogenic Signals (1)
Ctl

MBD2

-actin VEGF-R2 eNOS p-eNOS

siRNA:

**
*

-actin

VEGF-R2 eNOS p-eNOS

WT

Rao et al. Circulation, 2012

Mbd2-/-

**
*

Suppression of MBD2 Activates EC

Survival and Proangiogenic Signals (2)

ERK1/2 P-ERK1/2
Rao et al. Circulation, 2012

Ctl

MBD2

Fold change

ERK1/2p-ERK1/2

siRNA:

siRNA: Ctl

WT

Mbd2-/-

MBD2

Ctl

-actin

BCL-2

siRNA:

MBD2

Fold change

Suppression of MBD2 Activates EC

Survival and Proangiogenic Signals (3)

siRNA:

-actin

BCL2

WT

Rao et al. Circulation, 2012

Mbd2-/-

Ctl

MBD2

Suppression of MBD2 Activates EC

Survival and Proangiogenic Signals (4)
Ctl

MBD2

P38

Fold change

p-p38

siRNA:

P38

p-p38

siRNA:

Rao et al. Circulation, 2012

WT

Mbd2-/-

Ctl

MBD2

eNOS Synergizes with VEGF-R2 to Promote

Endothelial Survival and Angiogenesis (5)
VEFG-R blocker

VEGF
VEGFR2-Ab
SU1498
L-NAME
p-VEGFR2
VEGFR2
p-eNOS
eNOS
p-ERK
ERK
BCL-2
-actin

Rao et al. Circulation, 2012

+
-

+
+
-

+
+
-

+
+

Blockade of eNOS function by L-NAME

completely abolished the protective effect of
MBD2 on perfusion recovery
MBD2-/-

B6

MBD2-/-, L-NAME

B6, L-NAME

BS

AS

DAY 2

DAY4

DAY7

DAY14

*
BS: the day before ischemic surgery;
AS: the day after ischemic surgery

Rao et al. Circulation, 2012

Summary
1. It is likely that MBD2 directly affects the
expression of eNOS and VEGF-R2;
2. ERK1/2, p38 and BCL-2 were down stream
of eNOS and VEGF-R signaling, and
therefore, loss of MBD2 only indirectly
affects their activation;
3. eNOS plays a predominant role in
perfusion recovery, it also synergizes with
VEGFR-2 to enhance endothelial survival
and angiogenesis, and as a result,
blockade of endogenous eNOS completely
diminished the protective effect resulted

How does MBD2 regulate eNOS and VEGF-R2

Hypothesis:
Ischemic/hypoxic (oxidative stress) insult induces a
DNA methylation turnover which results in the
repression of eNOS and VEGF-R2 expression in favor
of endothelial pathologies;
Loss of MBD2 leads to unable deciphering the
information encoded by DNA methylation, which
provides protection by enhancing endothelial survival
and angiogenic signals

MBD2 Binds to Key Genes Essential for Endothelial Function

F3

F4 F5

eNOS

R1 R2 R3
-1500

F6

F7

R4 R5

-1000

F8

R6

F9

R7

-500

R8

F10

R9 R10

500

GC-enriched

F1

VEGF-R2

C
-1500

-1000

F2

R1
R2
-500

CpG island 1
-602 to -317

F9/R9

F2

F10/R10

F1

F3
R3
0

CpG island 2
-270 to +232

500

F3/R3

Inpu
t

MBD2
Ab

Inpu
t

MBD2
Ab

Inpu
t

MBD2
Ab

-actin
Ab

Genom Negati
ic DNA
ve
contro
l
Genom
ic DNA

-actin
Ab

-actin
Ab

Genom
ic DNA

Negati
ve
contro
l

Negati
ve
contro
l

800 bp

200 bp

Rao et al. Circulation, 2012

Sonicated DNA

Ischemic insult induces a DNA

methylation turnover in the eNOS
TS
gene+392
-72 -23
+39 +94 +150+195

FP

-109 -67

+1

+67 +127 +167

+301
RP

Methylation rate of CpGelements in eNOS 5fanking region (%)

NC1
NC2
NC3
NC4
NC5
NC6
NC7
NC8
NC9
NC10
IC1
IC2
IC3
IC4
IC5
IC6
IC7
IC8
IC9
IC10

FP: forward primer; RP: reverse primer; TS: transcription site

unmethylate
d

Rao et al. Circulation, 2012

methylate
d

Normal

Ischemic

MBD2 only selectively binds to

the Methylated eNOS promoter

Unmethylated-probe +

Lysates
Methylated Probe

Shifted band

Free probe

Rao et al. Circulation, 2012

MBD2 represses eNOS promoter

activity after binding to the
methylated CpG DNA

Rao et al. Circulation, 2012

5-azadC treatment completely abolished the

effect of MBD2 siRNA on eNOS and VEGF-R2
expression
AZA
-actin VEGFR2 eNOS

siRNA
Ctl

MBD2

Ctl

MBD2

Conclusion:
MBD2 regulation of eNOS and VEGF-R2
transcription is DNA methylation dependent

Rao et al. Circulation, 2012

Key question:

Does MBD2 regulate eNOS and VEGF-R2

transcription with endothelial
specificity?

MBD2 Repression of eNOS

Transcription Involves Chromatin
Remodeling and Is Confined to ECs
Ctl

MBD2

Ctl

-actin

eNOS

siRNA:

DMSO

DMSO

Ctl

TSA

MBD2

Ctl

eNOS

WT

-actin

MBD2

TSA

Mbd2-/-

WT

Spenocytes

-actin

DMSO

Rao et al. Circulation, 2012

MBD2

HeLa

eNOS

siRNA:

HUVECs

TSA

Mbd2-/-

Trichostatin A

2012

2012

Scientific Career

Luckiness!!

The Center for Biomedical Research

Acknowledgments

(Faculty & Staff)

(Graduate Students)

2014

2014

2014

2015

2015
Mohammed

2014

2015

2015

2015

2015

2015

2015

Funding support:

Decio Eizirik ULB

Piotr Kraj (GRU)
Zheng Dong (GRU)
Yunchao Su (GRU)
Zhiguang Zhou (Xiangya)
Xudong Xiang (Xiangya)
Weikuan Gu (UT)

2016

2014

2016

Collaborators

2015

2016

2016

2016

NSFC Key Project grants (81130014, 81530024)

NSFC Oversea Collaborative Grant (81428001)
Ministry of Science & Technology (2016YFC1305000)
Program for Changjiang Scholars and Innovative
Research Team in University (IRT_14R20)
EFSD-CDC/Lily Program
Chinese Tobacco Bureau