WT1基因对骨肉瘤细胞增殖凋亡和侵袭作用的实验研究

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

目的探讨WT1基因在骨肉瘤中的表达及其对细胞增殖、凋亡和侵袭的影响。方法体外培养人成骨细胞hFOB1.19、骨肉瘤细胞MG-63细胞,Western blot法观察WT1蛋白的表达情况。体外培养MG-63细胞,分为siRNA-1组、siRNA-2组、阴性对照组(NC)组、空白对照组,分别转染WT1 siRNA-1、WTI siRNA-2及NC链,空白对照只加入转染试剂。转染后采用实时荧光定量PCR(qPCR)检测siRNA-1组、siRNA-2组、NC组、空白对照组MG-63细胞WT1基因mRNA的表达情况,选择抑制效果好的siRNA-2组用于后续实验。将转染后的MG-63细胞分为siRNA-2组、NC组,CCK-8法检测细胞增殖情况,流式细胞技术检测细胞凋亡率,比色法检测Caspase-3活性,Transwell小室法检测细胞侵袭能力,Western blot法检测WT1、Bcl-2、MMP-2蛋白的表达水平。结果Western blot法检测结果显示,骨肉瘤MG-63细胞中WT1蛋白的相对表达量1.29±0.14,明显高于成骨细胞hFOB1.19的0.23±0.07,差异有统计学意义(t=6.603,P<0.01)。qPCR检测结果显示,骨肉瘤MG-63细胞转染后,siRNA-1组、siRNA-2组WT1 mRNA相对表达量均低于NC组和空白对照组,其中siRNA-2的抑制效果更显著,差异有统计学意义(F=12.470,P<0.05)。根据此结果选择siRNA-2进行后续实验。转染siRNA-2后,CCK-8法检测结果显示,siRNA-2组光密度(OD)值为0.63±0.10,明显低于NC组的1.04±0.09,差异有统计学意义(t=3.088,P<0.05);流式细胞技术检测结果显示,siRNA-2组MG-63细胞凋亡率为5.10%±0.41%,明显高于NC组的2.57%±0.10%,差异有统计学意义(t=4.014,P<0.05);比色法检测结果显示,siRNA-2组细胞Caspase-3的相对活性为2.74±0.29,明显高于NC组的1.07±0.13,差异有统计学意义(t=5.177,P<0.05);Transwell小室法检测结果显示,siRNA-2组侵袭细胞为(75.0±9.5)个,明显少于NC组的(118.0±8.6)个,差异有统计学意义(t=3.340,P<0.05);Western blot检测结果显示,siRNA-2组WT1、Bcl-2、MMP-2蛋白相对表达量均明显低于NC组,差异均有统计学意义(t=6.063、3.979、4.232,P值均<0.05)。结论WT1在骨肉瘤细胞中高表达,靶向下调其表达水平可以抑制骨肉瘤细胞增殖和侵袭能力,诱导细胞凋亡,这可能与Bcl-2和MMP-2表达下调有关。

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.