Akt1通过磷酸化泛素结合酶E2S增强胶质瘤放化疗抵抗的实验研究  

Akt1 enhances the chemoradiotherapy resistance of gliomas through phosphorylation of ubiquitin-conjugating enzyme 2S： An experimental study

作　　者：胡力[1] 沈红[1] 刘利[1] 谢春成[1] 王海洋[1] 刘琴芳[2] 翁长江[2] 林志国[1] 

机构地区：[1]哈尔滨医科大学附属第一医院神经外科,150001 [2]中国农业科学院哈尔滨兽医研究所

出　　处：《中华神经外科杂志》2018年第2期194-199,共6页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金（81571646）;国家国际科技合作专项（2014DFA31630）;高等学校博士学科点专项科研基金（20132307110029）

摘　　要：目的 探讨Akt1增强胶质瘤放、化疗抵抗的机制.方法 通过免疫荧光实验检测正常星形细胞和4种恶性胶质瘤细胞（U87、U251、SF767以及U373）中泛素结合酶E2S（UBE2S）的表达情况;通过Western blot实验检测PTEN基因突变型和野生型胶质瘤细胞中UBE2S的表达差异;构建激活型Akt1载体,分别与UBE2S野生型或T152位点突变型载体共表达,观察PTEN/Akt信号通路对UBE2S的作用,以及UBE2S过表达后对非同源末端连接复合物的影响;采用流式细胞仪检测稳定敲低UBE2S的U87胶质瘤细胞对放、化疗的敏感性.结果 与正常星形细胞相比,胶质瘤细胞高表达UBE2S蛋白;PTEN突变型的胶质瘤细胞UBE2S的表达较野生型更稳定.构建激活型Akt1磷酸化UBE2S的T152位点,可促进UBE2S的稳定表达.UBE2S与Ku70、Ku80相互作用能促进Ku70的表达（P =0.009）.免疫印迹结果显示,敲低UBE2S的表达后,Ku70、Ku80和DNA-PKcs的表达量显著降低（P〈0.001）.流式细胞检测结果显示,UBE2S敲低可增强胶质瘤对放、化疗的敏感性（P〈0.001）.结论 UBE2S在恶性胶质瘤中高表达;Akt1可磷酸化UBE2S的T152位点,从而增强UBE2S的稳定性;敲低UBE2S能降低非同源末端连接复合物介导的DNA修复效率,从而增强胶质瘤细胞对放、化疗的敏感性.Objective To explore the mechanism of Akt1-associated enhancement of the chemoradiotherapy resistance of gtiomas.Methods The immunofluorescence （IF） experiment was conducted to test the expression of ubiquitin-conjugating enzyme 2S （UBE2S） in the normal astrocytes and glioma cells （U87,U251,SF767 and U373）.The Western blot was used to assess the differences of UBE2S expression in PTEN mutant and wild type glioma cell lines.To explore the role of PTEN/Akt signal pathway in UBE2S functions and the effect of UBE2S on the non-homologous end joining （NHEJ） repair,a constitutively activated Akt1 （Myr-HA-Akt1） and the UBE2S wild type or UBE2S T152A mutant were coexpressed.The flow cytometry （FCM） was used to test in the chemoradiotherapy sensitivity of the stable UBE2S knockdown glioma cells.Results Compared with the normal astrocytes,UBE2S was overexpressed in the malignant glioma cells.The expression of UBE2S was more stable in the PTEN mutant glioma cell line.Akt1 phosphorylated UBE2S at T152,which promoted the stabilization of UBE2S.UBE2S interacted with Ku70 and Ku80,which increased the expression of Ku70 （P =0.009）.The results of Western blot showed that the expression of Ku70,Ku80 and DNA-PKcs decreased significantly after knockdown of UBE2S expression （P 〈 0.001）.The results of FCM showed that the knockdown of UBE2S could enhance the chemoradiotherapy sensitivity of gliomas （P 〈 0.001）.Conclusions UBE2S is highly expressed in the malignant glioma.Akt1 physically interacts with UBE2S and phosphorylates UBE2S at T152,which is critical for the stabilization of UBE2S.The knockdown of UBE2S expression could retard NHEJ-mediated DNA repair and thus increase the sensitivity of chemoradiotherapy.

关 键 词：神经胶质瘤 化放疗 泛素结合酶E2S PTEN/Akt信号通路 

分 类 号：R739.4[医药卫生—肿瘤]