STIL Mutations E863K And A1047D Found In Human NTDs Interfere Sonic Hedgehog Signaling Pathway And Inhibitioncell Proliferation

Objectives:In development of neural tube, STIL genes involved in Sonic Hedgehog signaling pathway, promote neural progenitor cell proliferation and survival. Stil-/- mice display multiple abnormalities, including midline and left/right axial defects, and die by embryonic day(E) 9.5. In part of human NTD sample observed the phenomenon of STIL rare mutations, we want to study the STIL rare mutations affect Sonic Hedgehog signaling pathway in the process of development, and STIL rare mutations associated with human NTD.Methods:Using human NTD samples, through the enrichment of high-throughput sequencing detects whether patients with neural tube defects STIL rare genetic mutation. We used SPSS16.0 and European Molecular Biology Laboratory(EMBI) and the National Center for Biotechnology Information(NCBI) rare mutations of bioinformatics analysis, screening and validation of rare mutations. Then build mutant recombinant plasmid and transfection HEK293 T cells, Immunofluorescence technique is used to determine the correct positioning of recombinant plasmid. Using western blot technology to detect STIL protein expression level; through immune co-precipitation method to detect the interaction between STIL and SUFU; Using real time PCR technique to detect STIL and target genes Gli1 transcriptional factor m RNA expression level; By CCK8 cell proliferation detection method detecting mutant effects on cell proliferation.Results:(1)Bioinformatics analysis showed that compared with control : there is 12.15%STIL rare genetic mutation in our NTD samples, among them, 2.54% with rare missense mutation(2)Using western blot technology and real time PCR technique to detect rare variants affect gene expression, among them, E863 K, A1047 D mutation proteins levels drop and E863 K m RNA expression level decreased.(3)Through detect the target genes Gli1 of Sonic Hedgehog signaling pathway m RNA level detection, speculation E863 K STIL gene mutation has weakened the STIL regulation of Sonic Hedgehog signaling pathway.(4)Using CCK8 method, detect the cell proliferation, found that E863 K, A1047 D mutation affecting HEK293 T cell proliferation ability.Conclusions:STIL mutations affect gene expression, which affects changes in protein function,may cause Sonic Hedgehog signaling pathway conduction disorders, affect Sonic Hedgehog signaling pathways downstream target genes transcription level is reduced,caused cells proliferation decreased in development of neural tube. The findings revealed that STIL rare mutations is the possibility of neural tube defects.