摘要
目的通过利用神经纤毛蛋白1(NRP1)不同表达水平的肺上皮细胞模型,检测电离辐射诱导上皮-间充质转化(EMT)标志物及相关转录因子表达变化,探讨NRP1对辐射诱导肺上皮细胞EMT发生中的作用。方法对人肺Ⅱ型上皮细胞(A549)构建NRP1过表达(NRP1high-A549)和NRP1敲低(NRP1^low-A549)细胞模型,同时对小鼠正常肺上皮细胞(MLE-12)构建siNRP1-MLE-12细胞模型并进行验证。对NRP1high-A549、NRP1^low-A549和siNRP1-MLE-12细胞模型分别进行单次10GyX射线照射,于照射后0、12、24和48h提取总蛋白和总RNA。利用Westernblot法和q-PCR法检测各组细胞模型中EMT标志物(β-catenin、N-cadherin和Vimentin)及相关转录因子(Twist和ZEB1)的变化。结果野生型A549和MLE-12细胞照射后NRP1mRNA和蛋白表达显著升高,间充质标志物(Vimentin和N-cadherin)显著升高(A549:t=2.917、7.361,P<0.01;MLE-12:t=9.652、31.357,P<0.01),ZEB1和Twist转录因子表达显著升高(A549:t=4.852、9.278,P<0.01;MLE-12:t=30.985、17.266,P<0.01)。NRP1^low-A549和siNRP1-MLE-12组受照后Vimentin和N-cadherin表达显著下降(NRP1^low-A549:t=10.077、15.707,P<0.01;siNRP1-MLE-12:t=5.745,P<0.01),上皮标志物(β-catenin)蛋白表达显著升高;而NRP1high-A549组照射后N-cadherin和Vimentin显著升高(t=16.055、5.560,P<0.01),而β-catenin显著下降。检测NRP1^low-A549组Twist转录因子在照射后显著下降(t=3.987,P<0.01),而NRP1high-A549组ZEB1和Twist转录因子照射后呈时间依赖性升高(t=11.289、2.903,P<0.01);siNRP1-MLE-12细胞模型照射后ZEB1转录因子显著下降(t=13.449,P<0.01),siNRP1-MLE-12组ZEB1和Twist蛋白表达均低于对照组,呈时间依赖性下降。结论NRP1可促进辐射诱导人和小鼠上皮细胞的EMT发生,而这种促进作用可能通过上调转录因子ZEB1和Twist的表达。
Objective To investigate the effect of neuropilin-1 (NRP1) on radiation-induced epithelial-mesenchymal transition (EMT) by measuring the expressions of EMT-related transcription factors in the irradiated cells with different levels of NRP1. Methods Human lung type Ⅱ epithelial cells (A549) were transfected with NRP1 over-expression lentiviral vector and NRP1 inhibition vector to construct two cell models of NRP1high-A549 and NRP1^low-A549. A NRP1 knock-down cell model was also constructed by transferring siNRP1 into normal mouse lung epithelial MLE-12 cells that was validated at both protein and mRNA levels. A single dose of 10 Gy X-ray was delivered to these cell models, then total protein and RNA were extracted at 0, 12, 24 and 48 h after irradiation. The expressions of EMT-related transcription factors (Twist and ZEB1) and EMT markers (β-catenin, N-cadherin, and Vimentin) in each cell model were detected by Western blot and qPCR. Results After 10 Gy irradiation, the expressions of NRP1 mRNA and protein were significantly increased in A549 and MLE-12 cells. The expressions of the mesenchymal markers (Vimentin and N-cadherin) and the transcription factors of ZEB1 and Twist were also significantly increased (A549: t=2.917, 7.361, 4.852, 9.278, P<0.01;MLE-12: t=9.652, 31.357, 30.985, 17.266, P <0.01). The expressions of Vimentin and N-cadherin were significantly decreased in NRP1^low-A549 (t=10.077, 15.707, P<0.01) and siNRP1-MLE-12 cells (t=5.745, P<0.01), but the expression of epithelial marker (β-catenin) was significantly increased in these cells. The expressions of N-Cadherin and Vimentin were significantly elevated (t=16.055, 5.560, P <0.01), while β-catenin decreased significantly in NRP1high-A549 cells. After irradiation, the transcription factor of Twist in NRP1^low-A549 group was significantly decreased (t=3.987, P<0.01), while the transcription factors of ZEB1 and Twist in the NRP1high-A549 group increased in a time-dependent manner (t=11.289, 2.903, P<0.01). After irradiation, the transcription fa
作者
陈志远
刘霖霖
魏威
董卓
吕亚慧
王蕊
衣峻萱
金顺子
Chen Zhiyuan;Liu Linlin;Wei Wei;Dong Zhuo;Lyu Yahui;Wang Rui;Yi Junxuan;Jin Shunzi(Key Laboratory of Radiobiology,National Health Commission,College of Public Health,Jilin University, Changchun 130021,China)
出处
《中华放射医学与防护杂志》
CAS
CSCD
北大核心
2019年第2期81-87,共7页
Chinese Journal of Radiological Medicine and Protection
基金
国家自然科学基金(81573085
81371890).
