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Xiu Liu Zhongyan Shan Weiping Teng Department of Endocrinology and Metabolism, First Affiliated Hospital of China Medical University. Effect of MiR-125b-5p on Thyroid Hormon es Depletion Cultured C17.2 Neural Stem Cell s. Background. Thyroid hormones and brain development. - PowerPoint PPT Presentation
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10th AOTA CONGRESS
Xiu Liu Zhongyan Shan Weiping Teng
Department of Endocrinology and Metabolism,
First Affiliated Hospital of China Medical University
Effect of MiR-125b-5p on Thyroid Hormones
Depletion Cultured C17.2 Neural Stem Cells
10th AOTA CONGRESS
Background
Thyroid hormones and brain development
• Thyroid hormones (THs) are essential for fetal neural development
• THs regulate the neuronal cytoarchitecture,neuronal growth and synaptogenesis
• The sensitive to the THs is not only remarked in the neonatal period but also prior to birth .
Ahmed OM. Int. J. Devl Neuroscience 26 (2008) 147–209
10th AOTA CONGRESS
MicroRNA effects on brain development
Neural inductio& Patterning
Let -7 Mir-9
Mir-430
Mir-29a/bMir-132Mir-134
Axon Extenison&
Synapse Formation
Migration
Mir-9Mir-134
Mir-9Mir-25
Mir-124
Proliferation
Mir-34aMir-128Mir-273
Differentiation
Background
10th AOTA CONGRESS
Background
Our previous work
MicroRNA expression profiling of rat offspring brains associated
with maternal hypothyroidism and subclinical hypothyroidism
during pregnancy in embryonic day 13
MiR-125b-5p
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Background
Depletion of L-3,5,3'-Triiodothyronine and L-Thyroxine in Euthyroid Calf Serum for Use in Cell Culture Studies of the Action of Thyroid Hormone
Regulation of Microglial Development: A Novel Role for Thyroid Hormone
Effect of thyroid hormone depletion on cultured murine cerebral cortex astrocytes
Endocrinology 1979,105: 80
Method used to study thyroid hormones deficency on cell lines
The Journal of Neuroscience, 2001, 21(6):2028–2038
Neuroscience Letters 2009,467:58–62
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Objective
• To identify the target gene of miR-125b-5p
• To investigate the effect of miR-125b-5p on thyroid hormones depletion cultured C17.2 neural stem cells
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Materials and Methods
http://www.targetscan.org/
mutant1 mutant2
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Materials and Methods
Relative dual-Luciferase activity
STAT3 3'UTR wt/mut-1/mut-2
Vectors and miR-125b-5p mimics co- transfected into HEK293 cells
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Materials and Methods
C17.2 neural stem cells
50nM miR-125b-5p mimics
50nM negative control
100nM miR-125b-5p mimics
100nM negative control
normal cultured thyroid hormonedepletion cultured
proliferationCell Counting
Kit-8
differentiationReal Time-
PCR:Tubb3,Gfap
target gene expression
WB:stat3,p-stat3
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Results
C17.2 neural stem cells nestin antibody and nuclei immune fluorescent staining
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Results
Dual-luciferase reporter system assay
0
1
2
3
4
5
6
7
stat3-wt stat3-mutant1 stat3-mutant2
miR-125 mimics
negative control
*p<0.05
**
10th AOTA CONGRESS
Results
Cell Counting Kit-8 proliferation assay
0.6
0.7
0.8
0.9
1
1.1
1.2
50nm-125 50nm-NC 100nm-125 100nm-NC
normal cultured
THs depletion cultured
*p<0.05
* *
10th AOTA CONGRESS
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
50nm-125 50nm-NC 100nm-125 100nm-NC
Tubb3
Results
Tubb3 and Gfap mRNA expression
0
0.2
0.4
0.6
0.8
1
50nm-125 50nm-NC 100nm-125 100nm-NC
GFAP
* *
*p<0.05
10th AOTA CONGRESS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
p-stat3 t-stat3
50nM-12550nM-NC100nM-125100nM-NC
Results
Total stat3 and phosphorylate stat3 protein expression
normal cultured THs depletion cultured
0
0.2
0.4
0.6
0.8
1
1.2
p-stat3 t-stat3
50nM-12550nM-NC100nM-125100nM-NC
* *
*p<0.05
10th AOTA CONGRESS
• STAT3 is one of miR-125b-5p target genes
• MiR-125b-5p may promote the differentiation of thyroid hormone depletion cultured C17.2 cell
Conclusion
10th AOTA CONGRESS