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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 10th AOTA CONGRESS 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 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 10th AOTA CONGRESS 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 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 10th AOTA CONGRESS Materials and Methods mutant1 http://www.targetscan.org/ mutant2 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 10th AOTA CONGRESS 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 10th AOTA CONGRESS Results C17.2 neural stem cells nestin antibody and nuclei immune fluorescent staining 10th AOTA CONGRESS Results Dual-luciferase reporter system assay * * *p<0.05 10th AOTA CONGRESS Results Cell Counting Kit-8 proliferation assay * * *p<0.05 10th AOTA CONGRESS Results Tubb3 and Gfap mRNA expression * * *p<0.05 10th AOTA CONGRESS 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 10th AOTA CONGRESS 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