Function Of-9247T/C Polymorphism And Expression Of The SOST Gene

Sclerostin (SOST) gene is mainly expressed in bone cells, which is located on the17q12-21. Sclerosis protein is a glycoprotein, which is a negative regulator of bone formation. Bone mineral density of SOST knockout mice increased significantly. SOST mutations can cause Van Buchem and Sclerosteosis characterized by hard-shrinking disease and a52kb deletion at SOST downstream35kb cause the Dutch van Buchem syndrome.We previously found that the SOST-9247T/C polymorphism was associated with BMD. Computational analysis shows that rs1230399is located in the center of the recognition domain of two collaborative transcription factors FOXA1and C/EBPα. Moreover, Tâ†'C mutation abolishes the binding of two transcription factor to the SOST gene. The prediction of bioinformatics need to be confirmed by experimental approaches.Experimental methods to study the interaction between DNA and protein include electrophoretic mobility of the shift assay, chromatin immunoprecipitation experiments, and reverse transcription-by polymerase chain reaction experiments.The purpose of this study is to study the influence of SNP rs1230399T/C polymorphism in the SOST expression and transcription factor FOXA1binding by EMSA and CHIP experiments. It is of great significance to understand the function of-9247T/C polymorphism and expression regulation of the SOST gene, and the pathogenesis of osteoporosis as well.Human osteosarcoma SAOS-2cell is used to study SOST gene expression. We first determined the FOXA1protein expression in SAOS-2by Western Blot. Then we studied the influence of rsl230399T/C polymorphism on SOST gene expression and transcription factor FOXA1binding by in vivo EMSA and in vitro CHIP. The EMSA results showed that the binding protein is not the FOXA1and C/EBPa protein as expected, and the T/C polymorphism have little influence on transcription factors binding. Besides, we did not get the DNA sequence of the transcription factor binding fragment in the further CHIP experiments.Basal expression of the SOST gene is studied by the RT-PCR method. The expression of the SOST gene is low in SAOS-2cells. The expression of the SOST and FOXA1genes decreased significantly after10μM and1μM estrogen treatment for24h, which is in accordance with higher prevalence of osteoporosis in postmenopausal women. Our result suggests that FOXA1may also be target gene of estrogen. Whether the significantly decreased SOST expression is caused by decreased FOXA1expression needs further study.