摘要
目的：使用小干扰RNA（siRNA）干扰人骨肉瘤MG-63细胞中营养缺乏自噬因子1（NAF1）的表达，观察对NAF1基因的沉默效果，并测定沉默后MG-63细胞生长增殖侵袭能力的变化。方法建立小干扰RNA（NAF1-siRNA）组、阴性对照（NC-siRNA）组、未行处理空白对照组。采用细胞划痕实验检测各组细胞迁移能力不同，采用Transwell法观察各组细胞侵袭能力变化，荧光素酶报告基因检测NF-κB信号通路的活性。结果与空白对照组相比，NAF1-siRNA干预MG-63细胞后，NAF1-siRNA组NAF1的mRNA和蛋白表达水平都明显降低（P〈0.01），MMP2蛋白表达水平降低，细胞迁移能力明显降低，NAF1-siRNA组穿过Transwell膜的细胞少于空白对照组，且NF-κB转录活性下降（P〈0.01）。结论通过siRNA技术降低NAF1表达可能通过抑制NF-κB通路，影响MMP2蛋白表达水平，抑制骨肉瘤MG-63细胞的迁移和侵袭能力。
Objective To investigate the gene-silencing effect of small interfering RNA（siRNA） on nutrient-deprivation autophagy factor-1（NAF1）in human osteosarcoma MG-63 cells transfected with the special siRNA,and to determine the biological behaviors of the transduced MG-63 cells,including invasion and metastasis. Methods The cultured MG-63 cells were divided into three groups including NAF1-siRNA group,negative control（NC-siRNA）group and the untreated blank control group. Wound healing assay was used to investigate the migration capability of the MG-63 cells from each group while the invasion potency was determined by transwell assay. Luciferase reporter gene assay were used to explore the activity of NF-κB pathway. Results NAF1 expression in MG-63 cells transfected with NAF1-siRNA was significantly down-regulated at both the mRNA and protein levels compared with normal MG-63 cells（P〈0.01）,accompanied with the reduced expression of MMP2 protein. The invasion and metastasis of MG-63 cells were obviously inhibited in NAF1-siRNA group compared with the control group（P〈0.01）. Furthermore,the transcription activity of NF-κB was down-regulated,and fewer cells could penetrate Transwell membrane when NAF1 was effectively inhibited by NAF1-siRNA, compared with the untreated blank control group （P〈0.01）. Conclusion Down-regulation of NAF1 by siRNA may inhibit the invasion and metastasis of osteosarcoma cells,probably mediated via the blockage of NF-κB signaling pathway and reduced expression of MMP2 at the protein level.
出处
《中华生物医学工程杂志》
CAS
2015年第2期130-133,共4页
Chinese Journal of Biomedical Engineering
基金
广州市卫生局一般引导项目支持（20121A011012）
关键词
骨肉瘤
RNA干扰
营养缺乏自噬因子1
Osteosarcoma
RNA interference
Nutrient-deprivation autophagy factor-1
