Abstract: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.
张翼, 李甲振, 张岩, 卢新昌, 张彬. WT1基因对骨肉瘤细胞增殖凋亡和侵袭作用的实验研究[J]. 中华解剖与临床杂志, 2019, 24(4): 341-346.
Zhang Yi, Li Jiazhen, Zhang Yan, Lu Xinchang, Zhang Bin. Effects of Wilms tumor 1 on the proliferation, apoptosis and invasion of osteosarcoma cells. Chinese Journal of Anatomy and Clinics, 2019, 24(4): 341-346.
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