Post on 30-Dec-2015
description
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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.
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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.
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
.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
10th AOTA CONGRESS
.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
10th AOTA CONGRESS
-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
10th AOTA CONGRESS
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
10th AOTA CONGRESS
.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
10th AOTA CONGRESS
.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
10th AOTA CONGRESS
.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