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10 th 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

Xiu Liu Zhongyan Shan Weiping Teng Department of Endocrinology and Metabolism,

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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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Page 1: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 2: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 3: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 4: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 5: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

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

Page 6: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

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

Page 7: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

Materials and Methods

http://www.targetscan.org/

mutant1 mutant2

Page 8: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

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

Page 9: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

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

Page 10: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

Results

C17.2 neural stem cells nestin antibody and nuclei immune fluorescent staining

Page 11: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS

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

**

Page 12: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

* *

Page 13: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 14: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 15: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

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

Page 16: Xiu Liu     Zhongyan Shan      Weiping Teng   Department of Endocrinology and Metabolism,

10th AOTA CONGRESS