PI3KI/Akt1在辐射诱导的乳腺癌细胞自噬发生中表达调控及作用机制

目的研究不同剂量X线诱导乳腺癌细胞株MCF-7自噬发生过程中Akt表达的变化以及探讨小分子干扰RNA沉默Akt基因后对自噬的影响及可能的作用机制。方法采用实时荧光定量PCR方法检测MCF-7中Akt基因表达,Western blot方法检测Akt蛋白表达;实时荧光定量PCR方法检测Akt过表达和沉默模型中MAP1LC3B基因表达。结果正常照射组MCF-7细胞量效研究表明,在4、8、16 h Akt1表达分别在24、、8Gy区间内呈剂量依赖下降变化,照射后Akt1总体表达均低于假照组(P0.01);32 h量效结果表明,Akt1表达明显呈剂量依赖下降变化,4、8、12Gy有显著性差异(P0.05)。时间效应研究表明,48、、12Gy时程研究Akt1表达量均于8 h降至最低,差异显著(P0.05),照射后Akt1基因总体变化均低于假照组。正常照射组MCF-7细胞Akt1蛋白表达情况为,照射后2 h Akt1蛋白表达即有明显下降趋势,24 h达至最低值;4Gy时效研究表明,照射后0.5 h Akt1蛋白表达明显下降,8 h达至最低值;8 Gy时效研究表明,照射后Akt1蛋白表达在0.5-4 h区间内呈时间依赖下降趋势,4 h达至最低值。Akt1 siRNA模型照射组量效研究表明,MAP1LC3B表达在8、163、2 h均有不同程度升高变化,12 Gy达至最大值(P0.05);时间效应关系表明,2、48、Gy MAP1LC3B表达在16 h3、2 h上升变化显著(P0.05),12 Gy时效表明,8、163、2 h MAP1LC3B表达呈剂量依赖上升变化,于32 h达至最大值(P0.05)。结论 X线可能通过抑制PI3KI/AKT转导通路活性来促进乳腺癌细胞株MCF-7发生自噬。

Changes of PI3KI/Akt1 expression in radiation-induced autophy in breast cancer cells

Objective To investigate the expression of AKT in breast cancer cell line MCF-7 in the process of autophagy induced by different doses of ionizing irradiation and the possible mechanism on autophagy after silencing AKT gene by siRNA.Methods Realtime RT-PCR was used to detect AKT mRNA expression,Western blot was used to detect the expression of AKT protein;The model of AKT gene overexpression and MAP1LC3B silencing were detected by Realtime quantitative PCR.Results Dose-response of MCF-7 irradiated groups studies had shown that dose-effect study of Akt1 mRNA expression changed in a dose-dependent decreasing at the 2-8 Gy interval in 4、8、16 h study（P〈0.01）,dose-effect of Akt1 expression in 32 h study was significantly decreased in a dose-dependent changes,there was a significant difference in 4,8,12 Gy（P〈0.05）.Respectively,time-response of MCF-7 cells studies had shown that dose-effect study of Akt1 mRNA expression had a significant difference decreased to peak valley at the 8 h in 4,8,12Gy study（P〈0.05）,overall of Akt1 genes changes were lower than control groups after irradiated.The proteins of MCF-7 irradiated groups of studies had shown that Akt1 proteins expression had a clear downward trend in 2 h timeeffect study,down to minimum at the 24 h.Akt1 proteins expression decreased significantly at 0.5 h in 4 Gy time-effect study,down to minimum at the 8 h .And its expression decreased in a dose-dependent changes trend at 8 Gy in 0.54 h time-effect study,down to minimum at the 4 h.Dose-response of Akt1 siRNA model irradiated groups of MCF-7 cells studies had shown that MAP1LC3B mRNA expression increased by varying levels in 8、16、32 h dose-effect study and up to peak at the 12 Gy（P〈0.05）.Respectively,time-response of MAP1LC3B of Akt1 siRNA model cells studies had shown that MAP1LC3B mRNA expression increased significantly at the 16,32 h study in 2,4,8Gy（P〈0.05）,and its expression increased in a dose-dependent at the 12 Gy in 8-32 h time-effect study,up to maximum at the 32 h（P〈0.05）.Conclusion These results strongly suggested that Akt1 plays an important role in irradiation-induced autophagy in breast cancer cell,and indicated that PI3KI/Akt pathway may serve as a theoretical basis of clinical treatment of breast cancer.