中华放射医学与防护杂志

陈志远,刘霖霖,魏威,等.神经纤毛蛋白1在辐射诱导肺上皮细胞转化中的调控作用[J].中华放射医学与防护杂志,2019,39(2):81-87.Chen Zhiyuan,Liu Linlin,Wei Wei,et al.Regulation of neuropilin-1 in radiation-induced transformation of lung epithelial cells[J].Chin J Radiol Med Prot,2019,39(2):81-87

神经纤毛蛋白1在辐射诱导肺上皮细胞转化中的调控作用

Regulation of neuropilin-1 in radiation-induced transformation of lung epithelial cells

投稿时间：2018-06-27

DOI：10.3760/cma.j.issn.0254-5098.2019.02.001

中文关键词: 神经纤毛蛋白1(NRP1) 电离辐射上皮-间充质转化(EMT) 转录因子

英文关键词:Neuropilin-1 Ionizing radiation Epithelial-mesenchymal transition Transcription factor

基金项目:国家自然科学基金（81573085； 81371890）

作者	单位	E-mail
陈志远	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
刘霖霖	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
魏威	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
董卓	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
吕亚慧	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
王蕊	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
衣峻萱	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021
金顺子	吉林大学公共卫生学院国家卫生健康委员会放射生物学重点实验室, 长春 130021	jinsz@jlu.edu.cn

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中文摘要:

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（NRP1^high-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NRP1^high-A549、NRP1^low-A549和siNRP1-MLE-12细胞模型分别进行单次10 Gy X射线照射，于照射后0、12、24和48 h提取总蛋白和总RNA。利用Western blot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NRP1 mRNA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NRP1^high-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NRP1^high-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。

英文摘要:

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 NRP1^high-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 NRP1^high-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 NRP1^high-A549 group increased in a time-dependent manner (t=11.289, 2.903, P<0.01). After irradiation, the transcription factor of ZEB1 decreased significantly in siNRP1-MLE-12 cells (t=13.449, P<0.01), and the protein expressions of ZEB1 and Twist in siNRP1-MLE-12 cells were lower than those of control group in a time-dependent manner. Conclusions NRP1 promotes radiation-induced EMT in human and mouse epithelial cells through up-regulation of transcription factors of ZEB1 and Twist.

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