印记基因PHLDA2过表达对骨肉瘤细胞的放射增敏作用及机制研究

Effects of the overexpression imprinted gene PHLDA2 on radiosensitivity of osteosarcoma

目的探讨印记基因PHLDA2过表达对骨肉瘤放射敏感性的影响及相关分子机制。方法通过基因转染技术，将真核表达质粒pEGFP—C3一PHLDA2导入骨肉瘤U20S细胞，经G418持续筛选获得稳定高表达PHLDA2蛋白的亚克隆细胞。实验分为3组：不转染的空白对照组，U20S；稳定转染pEGFP—C3质粒的阴性对照组，U20S—neo；pEGFP—C3-PHLDA2质粒的稳定转染组，U20S—PHLDA2。采用CKK8比色法测定4和8Gyx射线照射后细胞的增殖活性；克隆形成实验观察0～8Gy照射后细胞的存活能力；AnnexinV／PI染色法检测8Gy照射后的细胞凋亡；Westernblot测定蛋白的表达变化。建立人骨肉瘤裸鼠移植瘤模型，观察10Gy照射后PHLDA2基因的体内增敏效应。结果经筛选获得的骨肉瘤亚克隆U20S—PHLDA2细胞中PHLDA2蛋白表达上调（t=13．73，16．28，P〈0．05）。与U20S和U20S-neo组相比，PHLDA2高表达组在4和8Gy的增殖能力明显降低（t=5．00～8．23，P〈0．05），2～8Gy的克隆形成能力明显降低（t=2．52～-1．26，P〈0．05）；同时照后裸鼠移植瘤的生长抑制作用显著提高（t=3．27、2．91，P〈0．05）。8Gy照后，PHLDA2高表达组的凋亡率为（17．97±1．69）％，高于U20S组的（6．47±1．01）％和U20S—neo组的（7．15-t-O．96）％（t=10．11、9．61，P〈0．05）；同时Caspase-3活性明显提高（t=11．26、10．72，P〈0．05）。结论外源性PHDLA2基因在U20S细胞中稳定过表达能够提高骨肉瘤对x射线的敏感性，其机制可能是通过增强Caspase．3的活性、促进射线诱导的细胞凋亡作用实现的。

Objective To study the effects of PHLDA2 overexpression on radiosensitivity and the underlying mechanisms in human osteosareoma U2OS cell line. Methods To obtain the subclone, cells were exposed to G418 persistently after transfection of pEGFP-C3-PHLDA2 vector into U2OS cells. Three groups of blank control （ U20S ） , negative control （ U20S-neo ） and transfected group （ U20S-PHLDA2 ） were used. The expression of PHLDA2 in the subclone cells was determined by Western blot. After exposure to X-ray irradiation, cellular growth activity and survival were detected by CKK-8 assay and colony formation assay, respectively. The cell apoptosis was measured by the Annexin V/PI staining, and the apoptotic protein was analyzed by Western blot. The in-vivo effects of PHLDA2 on irradiation were evaluated by xenografts. Results Compared with U2OS group and U2OS-neogroup, the subclone cells were successfully obtained by G418 selection, in which the expression of PHLDA2 was upregulated（ t = 13.73,16.28, P 〈 0. 05）. In vitro, PHLDA2 overexpression significantly enhanced the response to radiation in U2OS cells with a reduction of colony survival and proliferation with the increase of doses （ t = 5.00 - 8.23 ,P 〈 0.05 ; t = - 2. 52 - - 1.26, P 〈 0. 05）. In vivo, PHLDA2-upregulated xenografts had more radiosensitivity than control groups with a significant inhibition of tumor growth （ t = 3.27,2.91, P 〈 0. 05 ）. After 8 Gy irradiation, the apoptosis was significantly increased （t = 10. 11,9.61, P 〈 0.05 ） , accompanied with the activation of Caspased-3 in U2OS-PHLDA2 cells, which was presented by upregulation of cleavedCaspase-3 （t = 11.26, 10.72, P 〈 0.05 ）. Conclusions Exogenetic expression of PHLDA2 could significantly enhance the radiosensitivity of human osteosarcoma cells, which may be attributed to the activation of Caspase-3 that increases irradiation-induced apoptosis.