降低B7-H3蛋白对受照肺癌细胞A549细胞周期和凋亡的影响

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。

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

Objective To 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.Results The 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.Conclusions Down-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.