| Characterized by low bone mineral density, osteoporosis is a systemic skeletal disease to lead to fracture easily, seriously affecting the public health. Differential gene expression profiling analyses and genome-wide association analysis in our laboratory found that STAT1 gene was closely related to osteoporosis. However, it was unclear for STAT1 gene to regulate in vivo bone mechanisms and development and to lead to phenotypic changes. So this study combined zebrafish model in vivo to further reveal molecular mechanisms of bone development. It has important significance for osteoporosis prevention, diagnosis, treatment and drug development. Genes and signaling pathways between zebrafish and human bone development are highly homologous. STAT1 gene is highly conservative, and zebrafish has two genes(stat1a and stat1b)related to human STAT1 which has two different isoforms STAT1-alpha and STAT1-beta. Interesting, stat1 a has high expression quantity in zebrafish embryos. Moreover, compared with other animal models,zebrafish has advantages such as small individual, transparent juvenile body, easy observation for bone development.We used the CRISPR/Cas9 targeting technology, and designed appropriate target sites on zebrafish stat1 a gene. And then specific sgRNA(final concentration of 20 μg/mL) and Cas9-mRNA(finalconcentration of 300 μg/mL) for in vitro synthesis was microinjected into zebrafish embryos. Also, activity assays confirmed the effectiveness of the selected sites. After extracted genomic DNA from zebrafish, T7E1 assays and Sanger sequencing successfully screened two zebrafish founder with stat1 a gene mutations. In the 20, 30 and 47 days after fertilization of F1 generation, randomly picked 10 zebrafish each of two mutant F1 generation and wild-type zebrafish for length measure, and found that mutant F1 generation grow faster than the wild-type. The results were consistent with the stat1-/- knockout mice found that increased bone mineral density and bone mass, suggesting that stat1 may affect skeleton development by inhibiting osteoblastogenesis. At present,homozygous mutant for stat1 a from F2 generation embryos was identified. Phyre software used to predict STAT1 protein spatial structure of the F1 generation mutant found some conserved domain sites of secondary structure changed. V638 and S639 of the STAT1 protein is a homodimer interface, but corresponding to these amino acid positions of F1 generation mutants, V630 and S631 is no longer homodimer interface.Gene stat1 a mutations may change the binding of STAT1 homodimer,and then affecting bone growth and development, leading to faster growth for zebrafish.Next, incubating zebrafish embryos with different concentrations of phosphate fludarabine(STAT1 inhibitor)for 10 days, we observed that themortality rate increased with the increase of the concentration. In less than 5 μg/mL concentration group, the mortality was about 10 %; in 10,20 and 40 μg/mL concentration group mortality reached 30 %- 40 %; and in 50 μg/mL concentrations group after five days with treatment of fludarabine, the mortality rate approached 70%. The experimental group has been found with slight or severe malformations after treatment with 3days. With increasing concentration, mortality of abnormal zebrafish increased, and the vast majority of dead fish were deformed. Fludarabine can inhibit expression of STAT1 which repress osteoblast differentiation.Deformity may be caused by physiological function imbalance of osteoclasts and osteoblasts, which generated by inhibition of STAT1,resulting skeletal dysplasia and ultimately producing changes in bone phenotype. |
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