电离辐射诱导乳腺癌细胞自噬相关基因DRAM表达的研究

Changes of DRAM expression in radiation-induced autophagy in breast cancer cells

目的研究电离辐射对乳腺癌细胞株MCF-7自噬相关基因DRAM表达变化的影响。方法采用GFP—LC3转染法检测自噬泡，实时荧光定量PCR方法检测DRAM基因表达，Westernblot方法检测LC3和DRAM蛋白的表达，采用磷酸钙共转染法构建DRAM沉默细胞模型。结果与假照组相比，8GyX射线照射后细胞中GFP—LC3表达升高；时间-效应关系表明，在8Gy照射后，自噬溶酶体蛋白LC3表达在2、4、8、16、24和32hi-升变化显著，各组均高于假照组（F=5．38、8．72、10．63、15．23、20．78和55．23，P〈0．05）；DRAM蛋白表达在8Gy照射后以时间依赖性增加，从2h开始上升，32h达至最高值（F=116．34，P〈0．05）。DRAM沉默细胞模型组中LC3和DRAM蛋白的表达明显低于假照组（F=20．36和40．35，P〈0．05）；对DRAM沉默细胞模型组进行8Gy照射后，DRAM蛋白表达与假照组相比虽有所增加，但仍低于8Gy照射组；DRAM沉默细胞模型组LC3蛋白的表达也低于照射组，沉默前后差异均有统计学意义（F=50．34，P〈0．05）。结论x射线可能通过激活DRAM基因来促进乳腺癌细胞株MCF-7发生自噬。

Objective To investigate the expression of DRAM in breast cancer cell line MCF-7 in radiation-induced autophagy. Methods GFP-LC3 transfection method was used to observe autophagy bubble. Real time-PCR was used to detect DRAM and MAPLC3 from transcriptional and translational level, respectively. The silencing vector from gene engineering was introduced by calcium phosphate transfection. Results Compared with the control group, GFP-LC3 increased significantly after 8 Gy irradiation by immunofluorescent assay, and the level of MAP-LC3 expression was higher than control group after 8 Gy irradiation by Western blot （ F = 5.38,8.72, 10. 63,15.23,20. 78 and 55.23, P 〈 0. 05 ）. DRAM protein expression increased significantly at 2 h in the 8 Gy time-dose study, up to maximum at the 32 h（F = 116.34,P 〈 0.05）. In DRAM model, the expression of LC3 and DRAM decreased significantly （F = 20. 36 and 40.35,P 〈 0.05 ） and DRAM expression increased 8 Gy post-irradiation, but still lower than that in 8 Gy irradiation wild-type group. The LC3 expression also decreasaed 8 Gy post-irradiation（F = 50. 34,P 〈 0. 05 ）. Conclusions DRAM plays an important role in irradiation-induced autophagy in breast cancer cell. DRAM might participate in the process and serve as a theoretical target for clinical treatment of breast cancer.