低氧诱导因子－1α去类泛素化修饰降低子宫内膜癌干细胞干性维持并增加对化疗敏感性的研究

Hypoxia inducible factor-1α deSUMOylation reduces the stemness maintenance ability of endometrialcancer stem cell and increases its chemosensitivity

目的通过诱导低氧诱导因子（HIF）－1α蛋白去类泛素化修饰降低子宫内膜癌干细胞的干性维持潜能，并增加其对化疗药物的敏感性。方法利用慢病毒质粒介导基因转染沉默KLE子宫内膜癌细胞泛素载体蛋白9（Ubc9）表达，免疫印迹方法检测Ubc9、小泛素相关修饰蛋白（SUMO）－1和HIF－1α蛋白表达水平；96孔板中培养并计算肿瘤干细胞克隆形成率及克隆球直径；流式细胞周期术检测细胞周期变化；MTT细胞毒性实验和流式细胞凋亡方法检测Ubc9沉默对子宫内膜癌干细胞对化疗药物顺铂的治疗敏感性。结果免疫印迹结果显示，Ubc9基因沉默效果良好，共价结合状态的SUMO－1及HIF－1α蛋白水平明显下降（P〈0.05）；Ubc9基因沉默使子宫内膜癌干细胞克隆形成率由（31.61±5.29）%下降至（11.42±3.07）%，同时细胞周期由G1向G2期后移，顺铂IC50由44.37 mg/L下降至7.39 mg/L，细胞凋亡率由（26.22±4.03）%下降至（41.59±5.37）%。结论通过沉默Ubc9基因表达能够诱导HIF－1α蛋白去SUMO化修饰，进而降低子宫内膜癌干细胞干性维持能力，并增强其化疗敏感性，为未来子宫内膜癌的基因治疗提供了新的参考和借鉴。

ObjectiveTo reduce the stemness maintenance ability of endometrial cancer stem cell and increase its sensitivity to chemotherapy by inducing hypoxia inducible factor 1α （HIF-1α） protein deSUMOylation.MethodsLentiviral plasmid mediated ubiquitin carrier protein 9 （Ubc9） gene silencing was transgened into KLE endometrial carcinoma cells. The expression of Ubc9, small ubiquitin-related modifier 1（SUMO1） and HIF-1α protein was detected by Western blotting. Then tumor stem cells clones were cultured in 96 well plates, and these clone balls diameter were calculated. Cell cycles were determined by flow cytometry. MTT cytotoxicity assay and flow cytometry method were used to test sensitivity of cisplatin to endometrial cancer stem cell.ResultsThe results of Western blotting showed that Ubc9 gene was silenced well, and the covalent binding state of SUMO-1 and HIF-1α protein levels were significantly decreased （P〈0.05）. Ubc9 gene silencing in endometrial cancer cells reduced clone formation rate by （31.61±5.29）% down to （11.42±3.07）%, while the cell cycle shift from G1 to G2. IC50 of cisplatin decreased from 44.37 mg/L to 7.39 mg/L, and the rate of cell apoptosis by （41.59±5.37）% down to （26.22±4.03）%.ConclusionThe stemness maintenance ability of endometrial cancer stem cell can be reduced through deSUMOylation of HIF-1α protein by silencing Ubc9 gene expression, and their sensitivity to chemotherapy be enhanced, which provides a new reference for future gene therapy of endometrial carcinoma.