OCT4过表达对神经母细胞瘤细胞顺铂敏感性的影响

Effects of OCT4 overexpression on cisplatin sensitivity in neuroblastoma cells

目的探讨OCT4过表达对SH—SY5Y细胞顺铂敏感性的影响及其作用机制。方法低黏附板无血清培养SH—SY5Y细胞，检测顺铂对SH—SY5Y细胞形成肿瘤球能力的影响，慢病毒转导OCT4建立sH—SY5Y细胞OCT4过表达株，Westernblot检测过表达OCT4后ABCG2蛋白水平的变化，MTT法检测SH-SY5Y细胞对顺铂的敏感性，PI染色流式细胞检测细胞凋亡率，罗丹明123检测细胞线粒体膜电位变化。结果5μmol／L顺铂连续孵育SH—SY5Y细胞4周后，细胞中OCT4mRNA和蛋白含量增高，细胞形成肿瘤球的数目明显提高，过表达OCT4可以在mRNA和蛋白水平明显上调SH—SY5Y中ABCG2的表达，20μmol／L顺铂作用72h细胞存活率，OCT4组为（69．65±4．20）％，Vector组为（25．75±3．27）％，OCT4过表达株对顺铂耐受能力显著提高（P〈0．01）。5μmol／L顺铂孵育72h，OCT4组细胞存活率为（1．5±0．69）％，Vector组为（12．88±4．35）％，OCT4过表达株细胞凋亡率较对照组明显减少（P〈0．01）。线粒体膜电位较对照组显著增高。结论过表达OCT4可以显著降低SH—SY5Y细胞对顺铂的敏感性，可能与0CT4上调ABCG2蛋白的表达有关。

Objective To explore the cisplatin sensitivity in SH-SYSY cells. Methods effects and mechanisms of OCT4 overexpression on The capacity of tumorsphere formation was measured by anchorage-independent serum-starved culture. OCT4 overexpression in SH-SY5Y cells was established by lentivirus transduction. The expressions of OCT4 and ABCG2 were analyzed by Western blot. Cisplatin sensitivity was assayed by methyl thiazolyl tetrazolium （MTT）. Cell apoptosis was detected by flow cytometry with PI staining. And rhodamine staining was used for determining mitochondrial membrane potential. Results The level of OCT4 expression increased after a 4-week incubation of 5 mol/L cisplatin. And tumorsphere formation was also elevated. The mRNA and protein expressions of AIN2G2 were up-regulated in OCT4 lentivirus-transduced cells versus controls. Cisplatin resistance was enhanced after OCT4 overexpression （viable cells after a 72-hour incubation of 20 mol/ L cisplatin, OCT4： 69. 65 ± 4. 20% vs vector.. 25.75 ± 3.27%, P〈0. 01）. The apoptotic rate was significantly lower in OCT4-transduced cells than controls after a 72-hour incubation of 5 mol/L cisplatin （OCT4：1.5 ± 0. 69% vs vector; 12,88 ± 4. 35%, P〈0. 01）. And mitochondrial membrane potential was higher in OCT4-transduced cells than that in controls. Conclusions OCT4 overexpression can significantly block cisplatin sensitivity potentially via a direct modulation of downstream target ABCG2 in SH-SY5Y cells.