Study On The Relationship Between GLI3 And Idiopathic Congenital Talipes Equinovarus And The Pathogenesis Mechanism

Study on the relationship between GLI3 and Idiopathic congenital talipes equinovarus and the pathogenesis mechanismIntroductionIdiopathic congenital talipes equinovarus (ICTEV) is a type of congenital limb deformity with an estimated incidence of 1-8%o live births. The mechanism by which ICTEV develops remains unclear even though the mechanical, neurological, muscular, bony, connective tissue, and vascular mechanisms have been proposed. Whilst both genetic and environmental factors are implicated, no specific genes have been identified. At present, investigations on human ICTEV mainly focus on the environmental factors at early stage of pregnancy and many syndromes with clubfoot malformations. Candidate genes and regions for ICTEV, such as COL9A1, CASP10, WNT7A, HOXD13 and GLI3 have been identified, respectively. However, little is known about the pathogenesis of human ICTEV.Our previous studies using the transmission disequilibrium test showed that the GLI3 gene maybe one of the predisposing genes in ICTEV. GLI family (GLI1, GLI2, and GLI3) is a highly conserved family. GLI3 is one of the signaling molecules in the zone of polarizing activity (ZPA). It limited the expression domain of some gene along the anterior-posterior (AP). Most GLI3 research focuses on digit abnormalities and deformities. GLI3 mutations cause limb development disorders about doigt. But it is still unknown whether GLI3 has relation with ICTEV. Some authors reported that HOXD13 has intimate relation with ICTEV. We further investigate the relation between GLI3 and ICTEV and how HOXD13 regulate GLI3. Samples:Veinous blood of 84 ICTEV patients and 15 ICTEV muscle tissues were obtained from the Department of Pediatric Orthopedic Surgery, Second Affiliated Hospital, China Medical University. Flexor hallucis longus and lung tissue were from nine normal cadavers provided by the institute of the forensic medicine of China medical university. Adult SD rats were obtained from the experimental animal center of our university. All procedures were carried out in accordance with an approved animal handling protocol.1. Mutation in the coding region of GLI3 in 84 ICTEV patients was detected by denaturing gradient electrophoresis.The total 10 exons (other 4 exons have been detected) of GLI3 gene were PCR from 84 ICTEV patients,20%-80%DGGE was used to detect the mutation in these 10 exons.2. The expression of GLI3 in the ICTEV patients'limb was detected by RT-PCRRNA was extracted from the flexor hallucis longus of ICTEV patients and normal controls and the lung of normal control, then tested the gene GLI3 expression.3. Make ICTEV model ratsThe ICTEV phenotype in rat embryos was induced by administration of 135 mg/kg all-trans-retinoic acid (ATRA) on gestation day 10 (GD10).4. The mRNA and protein levels of GLI3 were evaluated by Real Time-PCR and immunohistochemistry, Western Blotting, respectively.RNA and protein were extracted from the hindlimbs of the ICTEV model rat embryos and normal controls and detected GLI3 expression by Real Time PCR, Western Blotting and immunohistochemistry.5. P-Match software was used to analyze the sequence upstream of the transcription start site of the rat Gli3 gene.The 1100bp sequence uptream of rat GliS gene was obtained from http://www.ensembl.org, we used P-Match software to predict the binding sites of transcriptional factors on the sequence.6. Luciferase report vector assay.The promoter sequence in Gli3 were obtained by polymerase chain reaction, we constructed pGL3-Gli3 vectors. L6 cell were transfected pGL3-Gli3. After 24h, we analyzed the activity of Gli3 promotor.7. Chromatin immunoprecipitation assay.The chromatin was extracted from limb buds dissected from E14 wild-type embryos, sheared with an Enzymatic Shearing Kit to obtain 200～1000bp fragments. Gli3 antibody was used at the immunoprecipitation step. Eluted DNA from the sample and control was assessed for the presence of Gli3 DNA region by PCR.8. Electrophoretic mobility shift assay.Nucleoprotein was extracted from E14 rat embryonic limbs and incubated with the Gli3 upstream region-containingbinding site 2 labeled using a Biotin 3'End DNA labeling Kit for 60 min at room temperature in a gel shift buffer. Reactions were examined for nucleoprotein binding by electrophoretic mobility shift assays (EMSA) on a 10%nondenaturing polyacrylamide, transfered to the memberane, cross-linked. DNA binding bands were detected using a chemiluminescence system.9. Analysis Gli3 expression in L6 cell transfected Hoxd13 siRNAGli3 expression was analyzed after chemosynthesising Hoxd13 siRNA and transfecting it to L6 cell.10. Analysis Gli3 expression in L6 cell transfected HoxD13 expression vector. We constructed Hoxd13 expression vector, pcDNA-Hoxdl3, and transfected the expression vector in L6 cell. After 24h, the expression of Gli3 level was detected.Results1. No mutation was found in the exon 1～8,13 of GLI3 in 84 samples from patients with ICTEV.2. GLI3 is not expressed in the flexor hallucis longus of ICTEV patients and normal control.3. Expression of Gli3 in both mRNA and protein was higher in ICTEV model rat embryonic hindlimbs compared to normal control rat embryonic hindlimbs.4. The 5'region of the rat Gli3 gene contained two potential binding sites for the Hoxd13 protein designated Hoxd13 binding site 1(-667--663) and Hoxd13 binding site 2(-477--473).5. Hoxd13 binds with site 2 in Gli3 promoter region in the developing limb in rat.6. Hoxd13 directly binds to site 2 in Gli3 promoter region.Strong DNA binding was observed in the presence of the Hoxd13 protein. A competition experiment and supershift existence in the presence of the Hoxd13 antibody demonstrated the specificity of such binding. The result indicated Hoxd13 is bound to site 2 in vitro.7. Expression of Gli3 in mRNA was higher in L6 cell after Hoxd13 silence compared to control.8. The exogenous expression of Hoxdl3 down-regulated Gli3 transcription in L6 cells. Conclusions1. GLI3 gene mutation in coding region was not involved in outbreak in ICTEV.2. The decrease in HOXD13 expression led to an increase in the expression of GLI3, which maybe relate to ICTEV.3. Hoxdl3 directly controls the expression of Gli3 through Hoxd13 binding site 2 in the developing limb in rat embryo.