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

10 th 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

10 th AOTA CONGRESS MicroRNA effects on brain development Neural inductio& Patterning Let -7 Mir-9 Mir-430 Mir-29a/b Mir-132 Mir-134 Axon Extenison& Synapse Formation Migration Mir-9 Mir-134 Mir-9 Mir-25 Mir-124 Proliferation Mir-34a Mir-128 Mir-273 Differentiation Background

10 th 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

10 th 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

10 th 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

10 th AOTA CONGRESS Materials and Methods mutant1 mutant2

10 th 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 cell s

10 th AOTA CONGRESS Materials and Methods C17.2 neural stem cells C17.2 neural stem cells 50nM miR-125b- 5p mimics 50nM negative control 100nM miR-125b- 5p mimics 100nM negative control normal culturedthyroid hormone depletion cultured proliferation Cell Counting Kit-8 differentiation Real Time- PCR:Tubb3,Gfap target gene expression WB:stat3,p-stat3

10 th AOTA CONGRESS Results C17.2 neural stem cells nestin antibody and nuclei immune fluorescent staining

10 th AOTA CONGRESS Results Dual-luciferase reporter system assay *p<0.05 * *

10 th AOTA CONGRESS Results Cell Counting Kit-8 proliferation assay *p<0.05 * *

10 th AOTA CONGRESS Results Tubb3 and Gfap mRNA expression * * *p<0.05

10 th AOTA CONGRESS Results Total stat3 and phosphorylate stat3 protein expression normal cultured THs depletion cultured * * *p<0.05

10 th 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 cel l Conclusion

10 th AOTA CONGRESS