Preliminary Study Of Expression Regulation Of Nkx6.1 By Thyroid Hormone

Objective: Thyroid hormone is an important hormone secreted by the animal itself,It is a small, lipophilic tyrosine iodide. Importance of TSH for mammalian central nervous system development has been widely proved, Lack of TSH cells proliferation,differentiation, migration and mature will produce great influence, and ultimately affect the brain function. Homeobox gene NKx6.1(NK6 homeobox 1) first found expression in pancreatic ? cells, It was found that it played an important role in the differentiation and development of the central nervous system. Studies have shown that big rat pups Nkx6.1 gene expression levels will significantly change due to maternal low iodine diet. In this study thyroid hormone genomic features classic and non classic non-genomic features two aspects to the study of thyroid hormone on NKx6.1 homeobox gene regulatory mechanisms. For further research of genes Nkx6.1 in the specific mechanism of action of hypothyroidism model lay the foundation.Methods:1 This experiment using real-time quantitative PCR and Western Blotting technology on the carrier of astrocytes in vitro testing thyroid hormones affect NKx6.1 hox genes expression. After cells were cultured for 24 hours the culture of RPMI1640 medium without serum and double resistance for removing hormone treatment, The experiment group is Low hormone group: serum substitute and double anti RPMI1640 culture medium, Normal group: 5% serum and double anti RPMI1640 culture medium, High hormone groups: normal culture medium was added to a final concentration of10-7M T3 culture medium. Each process is divided into three time periods culture, Respectively 12 h, 24 h and 48 h. The total RNA and protein were extracted. The expression levels of NKx6.1 were detected by real-time quantitative PCR and Blotting Western.2 We cloned a 2.4 kb rat NKx6.1 promoter by PCR and predicted the potential transcription factor binding sits with bioinformatics method; three promoter segments with different length were obtained by promoter deletion analysis and cloned into reporter vecto. Co series of NKx6.1 promoter fragments and T3 R, then the promoter activities were-transfected a determined in transciently transfected Rat Astrocytes.3 The expression of p-ERK, p-AKT and Gli1 in high hormone cultured astrocytes was detected by Blotting Western with normal cultured astrocytes as control, To verify the activation of thyroid hormone on Shh,PI3 K / AKT and MAPK signaling pathway. Treatment of Shh, PI3K/AKT and MAPK signaling pathways with inhibitors, Using Blotting Western to detect the expression of NKx6.1 and Gli1, p-AKT and p-ERK after inhibition, To determine the cross-talking of Shh, PI3 K / AKT and MAPK signaling pathway.Results: 1Real time quantitative PCR and Blotting Western results showed that compared to the normal group, the low hormone group in 48 h expression was significantly down regulated, high hormone group in 12 h and 24 h expression up regulation, 48 h expression down.2 Amplification NKx6.1 first exon of the start codon of the 5'upstream 2325 bp promoter fragment.. According to the JASPAR online prediction T3 R transcription factor binding sites for truncated series, Successfully constructed three NKx6.1 sequence truncated promoter reporter vector pGL3-2.3, GL3-1.8,pGL3-1.5, The activation effect of pc DNA-T3 R on pGL3-2.3 and pGL3-1.8 is considerable, And a significant reduction in the activation of pGL3-1.5.3 Western Blotting showed that, High hormone cultured cells Gli1, p-AKT and p-ERK expression were increased. The expression of p-ERK decreased in the inhibition of MAPK signaling pathway, and there was no significant change in the expression of Gli1, p-AKT and NKx6.1, The expression of p-AKT decreased in the inhibition of PI3K/AKT signaling pathway, and there was no significant change in the expression of Gli1, p-ERK and NKx6.1, The expression of Gli1, p-AKT, p-ERK and NKx6.1decreased in the inhibition of Shh signaling pathway.Conclusion: 1 Thyroid hormone induces the expression of NKx6.1.2 the results of dual luciferase assay showed that the region of-1887bp~-1507 bp presented the highest activities, contained the key cis-regulatory element.3 Shh signaling pathways can affect the thyroid hormone in NKx6.1 gene expression regulation.