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低氧诱导因子-2α基因沉默对骨肉瘤细胞株MG-63生物学行为的影响

朱波1,李奇2   

  1. 1. 南方医科大学珠江医院骨科
    2.
  • 收稿日期:2013-11-25 修回日期:2014-03-17 出版日期:2014-08-15 发布日期:2014-08-15
  • 通讯作者: 朱波

The Effect of Hypoxia-inducible Factor(HIF)-2alpha silence on Osteosarcoma MG-63 cells

Bo ZHU 2   

  • Received:2013-11-25 Revised:2014-03-17 Published:2014-08-15 Online:2014-08-15
  • Contact: Bo ZHU

摘要: 【摘要】 目的   探讨低氧诱导因子-2(HIF-2α)在骨肉瘤形成过程中的作用以及HIF-2α基因沉默对低氧状态下骨肉瘤MG-63细胞的影响。 方法   小干扰RNA(siRNA)沉默HIF-2α基因,获得MG-63/siHIF-2α(siHIF-2α),阴性对照为MG-63/scramble(NC)细胞,分为siHIF-2α组和NC组,MTS试剂检测细胞活性,划痕迁移试验检测细胞迁移能力,Western Blot检测HIF-2α、VEGF、p-Erk1/2及Mcl-1的表达,集落形成试验和裸鼠皮下移植瘤试验证实HIF-2α基因沉默对肿瘤生长的影响。 结果   低氧诱导HIF-2α蛋白表达。低氧12h和24h,siHIF-2α组细胞活性均低于NC组(P=0.0211, 0.0017),siHIF-2α组相对划痕宽度均大于NC组(P=0.0024, <0.0001)。 1000-5000细胞种植数的siHIF-2α组的集落形成率均小于NC组(P=0.0027, 0.0038, 0.0063, 0.0276, 0.0062)。HIF-2α基因沉默抑制HIF-2α、VEGF、p-Erk1/2和Mcl-1的表达。裸鼠皮下移植瘤siHIF-2α组肿瘤体积和重量均小于NC组(P=0.006, <0.0001)。 结论   HIF-2α对骨肉瘤的存活、迁移及生长有促进作用,HIF-2α基因沉默可作为骨肉瘤的临床治疗的新策略。

关键词: 骨肉瘤, 细胞系, 肿瘤, RNA, 小分子干扰, 基因沉默, 肿瘤移植, 小鼠

Abstract:

[Abstract] Objective   To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63cells un?der hypoxia.Methods   HIF-2αexpression level in MG-63cells under hypoxia was determined by Western Blot. Small in?
terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble(NC) cells.The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1in MG-63, NC and si?HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as?sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo.Results   Hypoxia im?proved HIF-2αexpression in MG-63cells in a time-dependent manner (F=2037.412,P<0.001). HIF-2αexpression un?der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for12h and24h were lower than that in NC cells (P<0.05orP<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for12h and24h were larger than that in NC group (P<0.01orP<0.01). When cell counts reach 1000-5000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05orP<0.01). The expression of VEGF, pErk/Erk and Mcl-1protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2αgroup in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion   Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.

Key words: Osteosarcoma, cell line, tumor, RNA, small interfering, gene silencing, neoplasm transplantation, 小鼠