降低B7-H3蛋白对受照肺癌细胞A549细胞周期和凋亡的影响

Study on the effects of B7-H3 down-regulation on cell cycle and apoptosis of lung cancer A549 cells

  • 摘要:
    目的观察用小干扰RNA(siRNA)敲降B7-H3蛋白的表达对人肺癌细胞A549的细胞周期和凋亡的影响。
    方法培养人肺癌A549细胞,将B7-H3蛋白siRNA瞬时转染于A549细胞(称为siB7-H3转染组)。实验分为4组,即对照组、siB7-H3转染组、照射组、照射+siB7-H3转染组。采用137Cs γ射线一次性照射,照射剂量为4 Gy;用Western blotting法和实时定量PCR分别检测B7-H3蛋白和mRNA的表达;流式细胞仪检测细胞周期和细胞凋亡的改变。
    结果与对照组相比,siB7-H3转染组的B7-H3蛋白水平明显降低,mRNA表达量也明显低于对照组且差异有统计学意义(t=-4.222,P=0.013)。siB7-H3转染组细胞的G0/G1期细胞阻滞,S和G2/M期细胞数量减少;与对照组相比,照射组出现轻微的G0/G1期阻滞和明显的G2/M期细胞阻滞,照射+siB7-H3转染组的G0/G1、G2/M期均有明显的阻滞。照射后48 h,与对照组相比,照射组细胞的坏死率和凋亡率明显升高,siB7-H3转染组和照射+siB7-H3转染组的坏死率和凋亡率均无明显改变。
    结论降低肺癌A549细胞中B7-H3蛋白表达水平可明显增加辐射诱导的G0/G1期阻滞,从而提示B7-H3表达水平的改变可能通过调节G0/G1细胞周期检查点而对肺癌细胞放射敏感性产生重要的调节功能。

     

    Abstract:
    ObjectiveTo explore the effects of B7-H3 down-regulation on cell cycle and apoptosis in lung cancer A549 cells. Method Human lung cancer cell line A549 were cultured. siB7-H3 RNA which can specifically silence the expression of B7-H3 protein was transfected into A549 cells. 137Cs γ-ray irradiation was used with a single dose of 4 Gy. Experiments included control group, siB7-H3 transfected group, irradiation group, and irradiation+siB7-H3 transfected group. After transfected with siB7-H3, Western blotting and quantitative real-time PCR assays were used to detect the expression of B7-H3 protein and mRNA in A549 cells. The cell cycle and apoptosis of A549 cells following 4 Gy irradiation were detected by flow cytometry.
    ResultsThe protein and mRNA expression of B7-H3 were significantly decreased in A549 cells transfected with siB7-H3 compared with the control group, and the differences between the two groups were statistically significant(t=-4.222, P=0.013). siB7-H3 transfection induced significant effect on cell cycle with increase of G0/G1 phase arrest and reduction of S and G2/M phase population. A mild enhanced G0/G1 phase arrest and an obvious enhanced G2/M phase arrest of irradiation group were detected compared with the control group. An enhanced G0/G1 and G2/M phase arrest of irradiation+siB7-H3 transfected group were detected compared with the control group. Compared with the control group, the necrosis and apoptosis induction of the irradiated group significantly increased at 48 h after irradiation. However, No significant alterations of necrosis and apoptosis induction were observed between irradiation group and irradiation+ siB7-H3 transfected group.
    ConclusionsDown-regulation of B7-H3 can significantly elevated the G0/G1 arrest in A549 cells following radiation. This conclusion indicated that the alteration of B7-H3 expression could play a key role in the regulation of the radiosensitivity of lung cancer via mediating the G0/G1 check point.

     

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