Effects of Wilms tumor 1 on the proliferation, apoptosis and invasion of osteosarcoma cells

Objective To investigate the differential expression of Wilms tumor 1(WT1) gene and its effects on the proliferation, apoptosis and invasion against osteosarcoma cells. Methods The human osteoblast hFOB1.19 and osteosarcoma MG-63 cells were cultured in vitro, and then the expression of WT1 protein was detected by Western blot. After the validation of WT1 over-expression in osteosarcoma cells, siRNA-1, siRNA-2, negative control (NC) targeting WT1 mRNA was transfected into osteosarcoma MG-63 cells while the transfection reagent only as the blank group, then the relative WT1 gene expression was confirmed by quantitative Real-time PCR (qPCR) for each group. The siRNA-2 group was selected for the further investigation while the NC group was set as the control group. CCK-8 and flow cytometry (FCM) were used to detect the proliferation and apoptosis rate; the activity of Caspase-3 and invasion were examined by the colorimetric assay and Transwell invasion assay, respectively. The expression of WT1, Bcl-2 and MMP-2 were detected by Western blot. Results The results from western blot confirmed the WT1 protein was relatively over-expressed in osteosarcoma cells (1.29±0.14) than osteoblast (0.23±0.07), there were statistical differences(t=6.603, P<0.01). After different WT1 siRNA-1 and siRNA-2 transfection into MG-63 cell, relative expression of WT1 gene significantly decreased compared with the NC group and blank group, especially the inhibition effect of siRNA-2 was more significant(F=12.470, P<0.05). So the siRNA-2 and NC group were selected for further investigation. After the siRNA-2 and NC transfection, the optical density(OD) value of siRNA-2 group(0.63±0.10)was significantly decreased compared with the NC group(1.04±0.09), there were statistical differences(t=3.088, P<0.05). The FCM results suggested the apoptosis cell increased significantly in siRNA-2 group(2.57%±0.10%) compared with the NC group(5.10%±0.41%), there were statistical differences (t=4.014, P<0.05), while the Caspase-3 activity increased remarkably (1.07±0.13 vs 2.74±0.29, t=5.177, P<0.05). The Transwell invasion assays showed the invaded cells decreased significantly in siRNA-2 group(118.0±8.6)compared with the NC group(75.0±9.5), there were statistical differences (t=5.812, P<0.05). The results from the Western blots indicated the expressions of WT1, Bcl-2 and MMP-2 were significantly reduced after siRNA-2 transfection compared with the NC group, there were statistical differences(t=6.063, 3.979, 4.232, all P values<0.05). Conclusions WT1 is over-expressed in osteosarcoma cells and its down-regulation can inhibit the osteosarcoma cell proliferation, invasion and induce cell apoptosis which are probably related with the down-expression of Bcl-2 and MMP-2. Key words: Osteosarcoma; Wilms tumor 1; Proliferation; Apoptosis; Invasion