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The study of microRNAs change in Iodine-induced autoimmune

thyroiditis model of NOD.H-2h4 mice

ZHAO Shu-Jie, BI Mei, Fan Chen-Ling, LIU Miao, LIU Tong, LI Yu-Shu, SHAN Zhong-Yan, TENG Wei-Ping

The Endocrine Department of the First Affiliated Hospital of China Medical University,

the Endocrine Institute of China Medical University, the Liaoning Provincial Key Laboratory of Endocrine Diseases

10th AOTA CONGRESS

Autoimmune thyroiditis (AIT) is one of typical organ specific autoimmune diseases.

BACKGROUND

Patients with AIT are characterized by lymphocytes infiltration in thyroid gland, destruction of thyroid follicular cells, serum autoantibodies such as TPOAb and TgAb in serum, and hypofunction.

10th AOTA CONGRESS

In present opinion, immune cells (especially T

cells and B cells) abnormality is immunological

basis in AIT. Possible mechanisms contain:

BACKGROUND

Abnormal Th1/Th2

Functional deficiency of Ts 、 Treg

Abnormal Th17

Cloned proliferation of autoreactive T cells

and B cells

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FITC-CD4

SS

C-H

AP

C-C

D2

5

AP

C-C

D2

5

PE-Foxp3 PE-Foxp3

Control 7.98% AIT 3.16%

BACKGROUND

Our previous study: CD4+CD25+Foxp3+

Treg cells in spleens from iodine-treated

NOD.H-2h4 mice and controls.

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FITC-CD4 FITC-CD4

PE

-IL

-17

PE

-IL

-17

Control 0.89% AIT 2.45%

BACKGROUND

IL-17 expression in splenocytes from iodine-treated

NOD.H-2h4 mice and controls.

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结果 5

0

0. 5

1

1. 5

2

2. 5

3

AI T Control

Foxp

3 mR

NA

#

0

0. 01

0. 02

0. 03

AI T Control

IL-1

7 mR

NA

#

0

0. 2

0. 4

0. 6

0. 8

AI T Control

ROR

rt m

RNA

#

0

0. 5

1

1. 5

2

2. 5

3

AI T Control

IL-1

0 mR

NA

#

#:P<0.05

BACKGROUND

Foxp3, IL-17 mRNA expression in splenocytes from

iodine-treated NOD.H-2h4 mice and controls.

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MicroRNAs (miRNAs) have recently emerged as a

major class of gene expression regulators linked to

most biological functions.

Several miRNAs have been identified as important

regulators for immune cell development, as well as

immune response.

BACKGROUND

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Recent studies have demonstrated that miR-155 plays a crucial

role in the function of pathogenic immune cells, including Th1

cells, Treg cells, B cells, DCs and Th17 cells.

BACKGROUND

O’connell RM. Nat Reviews Immunol 2010,22: 111-122

10th AOTA CONGRESS

It was reported that miR-326 is critical in regulating TH-17 differentiation and hence contributes to the pathogenesis of multiple sclerosis.

Knockdown or overexpression of miR-326 alleviated or aggravated EAE, respectively.

Martin AJ. Nat Immunol 2009,10: 1229-1231

BACKGROUND

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BACKGROUND

MiR-146a is among the miRNAs prevalently expressed in Treg cells and showed that it is critical for Treg functions.

ZHOU L. Cell & Molecul Biological 2011,8: 380-381

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OBJECTIVES

The aim of this study was to observe the

expression of miRNA-155, miRNA-146 and

miRNA-326 in splenic CD4+ T cells and thyroid

tissue of NOD.H-2h4 mice with iodine-induced

autoimmune thyroiditis.

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MATERIAL AND METHOD

0.05% NaI solution（ 12week ）

sterile water（ 12week ）

0.05% NaI solution（ 20week ）

sterile water（ 20week ）

iodine-treated groups （ n=10 ）

control group（ n=6 ）

iodine-treated groups （ n=10 ）

control group（ n=8 ）

get splenicCD4+T cells

get thyroid tissue

randomly divided

NOD.H-2h4

female mice at 4 weeks

of age

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Mice were sacrificed at the time point of 12 weeks after the beginning of experiment.

CD4+T cells were be purified by MACS

routinely separatemononuclear cells

stained with multicolor immunofluorescenceAnd detected by flowcytometry to analysis the

percentage of CD4+CD3e+cells

get spleen

Target micoRNA Expression of splenic CD4+T cells were

Measured by Real-time RT-PCR.

The severity of lymphocytic infiltration was observed using

HE-stained thyroid sections.

get thyroid

MATERIAL AND METHOD

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Mice were sacrificed at the time point of 20 weeks

after the beginning of experiment.

get another thyoid

Target micoRNA expressionIn thyroid were measured

by Real-time RT-PCR.

The severity of lymphocytic infiltration was observed using

HE-stained thyroid sections.

get thyroid

homogenete

MATERIAL AND METHOD

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Table.1 The rate of thyroiditis of thyroiditis after NOD-2h4 mice were feed with sterile water and iodine water at the time point of 12 and 20 weeks （ % ）

12 weeks 20 weeks Control 1000HI Control 1000HI

The rate of thyroiditis 0(0/10) 100# (6/6) 0 (0/10) 100# (8/8)

#P < 0.01 ， 1000HI ： 0.05% NaI solution group(about 1000 times of normal daily iodine intake amount, 1000HI)

Lymphocytic infiltration was observed in NOD.H-2h4 mice fed with high iodine water for 12 weeks and 20 weeks (P < 0.01).

RESULTS

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Fig.1 HE-stained thyroid sections after NOD.H-2h4 mice were fed with sterile water. (100X and 200X)

RESULTS

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Fig.2 HE-stained thyroid sections after NOD.H-2h4 mice were fed with 0.05%NaI for 12week . (100X and 200X)

RESULTS

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Fig.3 HE-stained thyroid sections after NOD.H-2h4 mice were fed with 0.05%NaI for 20 weeks . (100X and 200X)

RESULTS

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0.41%

FITC-CD4

PE-CD3e 79.95%

Fig.4 Splenic CD4+ Tcell separation ratio was analyzed by Immunofluorescence stain flow cytometric analysis. CD4+ Tcell (left) and rest of cells(right).

RESULTS

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group number mean value SD SE

microRNA 155 iodine-treated 10 4.974 4.324 1.367

control 6 1.418 0.9665 0.3946

microRNA 146 iodine-treated 10 3.332 3.085 0.9775

control 6 0.965 0.9347 0.3816

microRNA 326 iodine-treated 10 2.534 2.666 0.8431

control 6 1.039 0.3910 0.1596

t df P

Relative microRNA 155 2.499 10.431 0.031

Relative microRNA 146 2.260 11.481 0.044

Relative microRNA 326 1.742 9.635 0.113

Table.2 12 weeks after the beginning of experiment. miRNA-155, miRNA-146, miRNA-326 in splenic CD4+ Tcells were quantified by realtime quantitative PCR.

RESULTS

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.000000

1.000000

2.000000

3.000000

4.000000

5.000000

6.000000

7.000000

8.000000

9.000000

10.000000

1000HI control

P=0.031

miR

NA

-155exp

ress

ion

After 12th week, compared with control group （ 1.418±0.9665 ） , mice with AIT （ 4.974±4.324 ） have rised expression of miRNA-155 in splenic CD4+T cells (P < 0.05)

RESULTS

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.000000

1.000000

2.000000

3.000000

4.000000

5.000000

6.000000

7.000000

1000HI control

P=0.044

miR

NA

-14

6 e

xpre

ssio

n

After 12th week, compared with control group （ 0.9650±0.9347 ） , mice with AIT （ 3.332±3.085 ） have rised expression of miRNA-146 in splenic CD4+T cells (P < 0.05)

RESULTS

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-1.000000

.000000

1.000000

2.000000

3.000000

4.000000

5.000000

6.000000

1000HI control

p=0.113

miR

NA

-326 e

xpre

ssio

n

After 12th week, compared with control group （ 1.039±0.3910 ） , mice with AIT （ 2.534±2.666 ）， there is no obvious change in expression of miRNA-326 in splenic CD4+T cells (P > 0.05)

RESULTS

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group number mean SD SE

microRNA 155 iodine-treated 10 0.6929 0.2820 0.0892

control 8 0.1781 0.1234 0.0436

microRNA 146 iodine-treated 10 0.9529 0.2619 0.0828

control 8 0.2656 0.2350 0.0831

microRNA 326 iodine-treated 10 1.585 0.4035 0.1276

control 8 0.5103 0.1898 0.0671

t df P

Relative microRNA 155 4.786 16 0.000

Relative microRNA 146 5.859 15.737 0.000

Relative microRNA 326 7.456 13.353 0.000

Table.3 20 weeks after the beginning of experiment. miRNA-155, miRNA-146, miRNA-326 in thyroid tissues were quantified by realtime quantitative PCR.

RESULTS

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.000000

.200000

.400000

.600000

.800000

1.000000

1.200000

1000HI control

P=0.000

miR

NA

-155 e

xpre

ssio

n

After 20th week, compared with control group （ 0.1781±0.1234 ） , mice with AIT （ 0.6929±0.2821 ） have rised expression of miRNA-155 in thyroid tissues (P < 0.001)

RESULTS

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.000000

.200000

.400000

.600000

.800000

1.000000

1.200000

1.400000

1000HI control

P=0.000

miR

NA

-14

6 e

xp

ress

ion

After 20th week, compared with control group （ 0.2656±0.2350 ） , mice with AIT （ 0.9529±0.2619 ） have rised expression of miRNA-146 in thyroid tissues (P < 0.001)

RESULTS

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.000000

.500000

1.000000

1.500000

2.000000

2.500000

1000HI control

P=0.000

miR

NA

-32

6 e

xpre

ssio

n

After 20th week, compared with control group （ 0.5103±0.1898 ） , mice with AIT （ 1.585±0.4035 ） have rised expression of miRNA-326 in thyroid tissues (P < 0.001)

RESULTS

10th AOTA CONGRESS

Expression of miRNA-155, miRNA-146 and miRNA-326 were higher in both splenic CD4+ T cells and thyroid tissue of NOD.H-2h4 mice with iodine-induced autoimmune thyroiditis than in control mice. miRNA-155, miRNA-146 and miRNA-326 may play important role in the pathogenesis of AIT.

Further study are needed for over-expression or down-regulation of those microRNAs in AIT.

CONCLUSIONS

10th AOTA CONGRESS