多配体蛋白聚糖-1调控骨肉瘤细胞增殖和侵袭能力及机制研究
Effects of syndecan-1 on proliferation and invasion against osteosarcoma cell and its mechanisms
张翼 1张彬 1张岩 1卢新昌 1刘金磊 1李甲振1
作者信息
- 1. 郑州大学第一附属医院骨科,郑州 450052
- 折叠
摘要
目的 观察多配体蛋白聚糖-1(SDC1)调控骨肉瘤细胞增殖及侵袭能力并探讨其机制.方法 通过SDC1小干扰RNA转染骨肉瘤MG-63细胞,构建SDC1下调表达模型为实验组,以转染阴性对照链作为对照组,实时定量聚合酶链反应法验证SDC1 mRNA表达水平;比色法检测细胞增殖率和含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)活性变化;流式细胞技术检测细胞凋亡水平变化;细胞体外侵袭试验检测细胞侵袭能力变化;蛋白质印迹法(Western blot)实验检测SDC1和基质金属蛋白酶-9(MMP-9)蛋白表达水平.组间比较采用独立样本t检验.结果 SDC1 siRNA转染后,MG-63细胞中SDC1 mRNA相对表达水平低于对照组(0.52±0.15比1.06±0.07,t=5.650,P＜0.01);转染后24 h及48 h后siRNA组细胞增殖率均低于对照组(0.72±0.13比1.14±0.09、0.59±0.10 比 1.08±0.18,t=4.382、4.122,P＜0.01);流式细胞检测结果显示 siRNA组细胞凋亡率高丁对照组[(9.53±1.97)％比(4.58±1.09)％,t=3.808,P＜0.05],而 siRNA 组Caspase-3相对活性高于对照组(1.96±0.27比1.05±0.19,t=4.774,P＜0.01);体外侵袭实验证实siRNA组每视野下平均细胞数低于对照组[(76.22±12.02)个 比(112.40±18.30)个,t=3.695,P＜0.01];Western blot实验证实siRNA组细胞SDC1蛋白相对表达量低于对照组(0.56±0.24比1.07±0.10,t=3.411,P＜0.05);SDC1组细胞MMP-9蛋白相对表达量低于对照组,差异有统计学意义(0.62±0.11比0.97±0.14,t=3.405,P＜0.05).结论 靶向下调SDC1表达可以抑制骨肉瘤细胞增殖和侵袭能力,这与Caspase-3激活和下调MMP-9表达相关.
Abstract
Objective To investigate the effects of syndecan-1 on the proliferation and invasion a-gainst osteosarcoma cell and its mechanisms.Methods The expression of syndecan-1(SDC1)in osteosar-coma MG-63 cells was detected by Real-time quantitative polymerase chain reaction after SDC1 small inter-fering RNA(siRNA)transfection as the experimental group while the negative control transfection was set as the control group.After the validation of SDC1 mRNA down-expression,the colorimetric assays were used to detect the proliferation and cysteinyl aspartate specific proteinase-3(Caspase-3)activity;The apop-tosis rate was detected by the flow cytometry assays.The Transwell assay was used to determine the inva-sion activity.Then the expression levels of SDC1 and matrix metalloprotein(MMP-9)were detected by Western blotting respectively.Independent sample t-test was used for comparison between two groups.Results After SDC1 siRNA transfection to the OS MG-63 cells,relative expression of SDC1 mRNA in siRNA group was significantly less than the negative-control(NC)group(0.52±0.15 vs.1.06±0.07,t=5.650,P＜0.01).The proliferation at 24 h and 48 h in SDC1 siRNA group were lower than the NC group(0.72±0.13 vs.1.14±0.09,0.59±0.10 vs.1.08±0.18,t=4.382,4.122,P＜0.01).Flow cytometry assay showed the apoptosis rate in siRNA group is higher than the NC group[(9.53±1.97)％vs.(4.58±1.09)％,t=3.808,P＜0.05],while the relative activity of Caspase-3 was higher than NC group(1.96±0.27 vs.1.05±0.19,t=4.774,P＜0.01).Transwell assay showed the invaded cells in siRNA group were significantly less than the NC group[(76.22±12.02)cells vs.(112.40±18.30)cells,t=3.695,P＜0.01].After the transfection,the results from the Western-blot showed that the relative ex-pression of SDC1 in siRNA group was lower than the NC group(0.56±0.24 vs.1.07±0.10,t=3.411,P＜0.05),while the relative levels of MMP-9 in siRNA group was less than NC group(0.62±0.11 vs.0.97±0.14,t=3.405,P＜0.05).Conclusion Down-regulation of SDC1 inhibited the proliferation,invasion of osteosarcoma MG-63 cells which was probably related with the Caspase-3 activation and the in-hibition of MMP-9 expression.
关键词
多配体蛋白聚糖-1/骨肉瘤/增殖/凋亡/侵袭Key words
Syndecan-1/Osteosarcoma/Proliferation/Apoptosis/Invasion引用本文复制引用
出版年
2024
NETL
NSTL
万方数据