Effect of Histone Deacetylase Inhibitor to Osteosarcoma through Specificity Protein 1/ Phospholipase D1 Pathway

Abstract

To study the effect of histone deacetylase inhibitor to osteosarcoma by targeting specificity protein 1 and regulating Phospholipase D1. Human osteosarcoma cell line MG63 cells were cultured in Dulbecco’s Modified Eagle’s medium medium containing penicillin (100 U/ml) and 10 % fetal bovine serum at 37°. siRNA-specificity protein 1 and psiCHECK-Phospholipase-WT plasmid were constructed and transfected into MG63 cells. Dual luciferase reporter gene assay were conducted to verified the combination site of specificity protein 1 and Phospholipase. MG63 cells were treated with tumor-specific antigen (5 g/ml) or PBS (negative control) for 48 h at 37°. Western blot was used to test the phospholipase D1, nuclear specificity protein 1 and cytoplasmic specificity protein 1 in MG63 cells. Cell Counting Kit-8 and clone formation assay were conducted to analyze the difference in cell viability and proliferation of tumorspecific antigen -treated MG63 cells and tumor-specific antigen free cells. Western blot result showed that nuclear and cytoplasmic specificity protein 1 in tumor-specific antigen treated MG63 cells both decreased dramatically compared to tumor-specific antigen free cells (p<0.05). Phospholipase in tumor-specific antigen treated MG63 cells also reduced (p<0.05). Fluorescence analysis showed that the ratio of Firefly luciferase to Renilla luciferase in MG63 cells transfected with psiCHECK-Phospholipase-WT plasmid and si-specificity protein 1 was significantly lower than that of si-NC (p<0.05), while the ratio of firefly luciferase to Renilla luciferase in MG63 cells transfected with psiCHECK-Phospholipase-WT plasmid and si-specificity protein 1 had no significant difference with si-NC control (p<0.05). After tumor-specific antigen treatment for 24 h, the proliferation rate of MG63 cells was (65.20 %±2.31 %), which was significantly lower than that of PBS control group (96.63%±1.14%) (p<0.05) by CCK-8 method. Cell formation assay showed that the amount of MG63 cells treated with tumor-specific antigen decreased significantly (p<0.05). The gene expression of specificity protein 1 and Phospholipase in tumor-specific antigen -treated MG63 cells decreased significantly compared with tumor-specific antigen free cells (p<0.05). The expression of survivin in tumor-specific antigen -treated MG63 cells were significantly lower than that in tumor-specific antigen free cells (p<0.05). The gene expression levels of p21 in tumor-specific antigen -treated MG63 cells were significantly higher than that in tumor-specific antigen free cells (p<0.05). The expression of cyclin A2 had no significant difference between two groups (p<0.05). Tumor-specific antigen can increase the early apoptosis rate, late apoptosis rate and total apoptosis rate of MG63 cells compared to the non- tumorspecific antigen -treatment cells (p<0.05). Specificity protein 1 could bind and target with Phospholipase promotor/enhancer region. Histone deacetylase inhibitor can reduce the proliferation and apoptosis of osteosarcoma cells through decreasing the expression of Phospholipase by inhibiting the protein expression and nuclear migration of specificity protein 1).