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Effect of MiR-125b-5p on Thyroid Hormones
Depletion Cultured C17.2 Neural Stem Cells
Xiu Liu
Zhongyan Shan
Weiping Teng
Department of Endocrinology and Metabolism,
First Affiliated Hospital of China Medical University
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
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Background
MicroRNA effects on brain development
Neural
inductio&
Patterning
Let -7
Mir-9
Mir-430
Proliferation
Mir-9
Mir-25
Mir-124
Migration
Mir-9
Mir-134
Differentiation
Mir-34a
Mir-128
Mir-273
Axon
Extenison&
Synapse
Formation
Mir-29a/b
Mir-132
Mir-134
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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
Method used to study thyroid hormones deficency on cell lines
 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
Endocrinology 1979,105: 80
 Regulation of Microglial Development: A Novel Role for
Thyroid Hormone
The Journal of Neuroscience, 2001, 21(6):2028–2038
 Effect of thyroid hormone depletion on cultured murine
cerebral cortex astrocytes
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
mutant1
http://www.targetscan.org/
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
normal cultured
50nM miR-125b5p mimics
proliferation
Cell Counting
Kit-8
50nM negative
control
thyroid hormone
depletion cultured
100nM miR-125b5p mimics
differentiation
Real TimePCR:Tubb3,Gfap
100nM negative
control
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
*
*
*p<0.05
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Results
Cell Counting Kit-8 proliferation assay
*
*
*p<0.05
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Results
Tubb3 and Gfap mRNA expression
*
*
*p<0.05
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Results
Total stat3 and phosphorylate stat3 protein expression
normal cultured
THs depletion cultured
1.4
1.2
1.2
1
0.8
1
*
50nM-125
50nM-NC
100nM-125
100nM-NC
0.6
0.4
0.2
0.8
*
50nM-125
50nM-NC
100nM-125
100nM-NC
0.6
0.4
0.2
0
p-stat3
t-stat3
0
p-stat3
t-stat3
*p<0.05
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Conclusion
• STAT3 is one of miR-125b-5p target genes
• MiR-125b-5p may promote the differentiation of
thyroid hormone depletion cultured C17.2 cell
10th AOTA CONGRESS
10th AOTA CONGRESS