| 刘畅,彭超君,王利利,朱巍,徐加英,焦旸.抑制长链非编码RNA-BG对人正常肺上皮细胞系Beas-2B放射敏感性的影响[J].中华放射医学与防护杂志,2014,34(5):323-328 |
| 抑制长链非编码RNA-BG对人正常肺上皮细胞系Beas-2B放射敏感性的影响 |
| Effect of suppression of long non-coding RNA-BG on radiosensitivity of normal human bronchial epithelial cell line Beas-2B |
| 投稿时间：2013-11-20 |
| DOI：10.3760/cma.j.issn.0254-5098.2014.05.002 |
| 中文关键词: 长链非编码RNA siRNA 电离辐射 细胞周期 DNA损伤响应 |
| 英文关键词:LncRNA siRNA Ionizing radiation Cell cycle DNA damage response |
| 基金项目:国家自然科学基金（81302382）；江苏高校优势学科建设工程资助项目（PAPD） |
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| 中文摘要: |
| 目的 探讨长链非编码RNA-BG对人正常肺上皮细胞Beas-2B放射敏感性的影响。方法 设计合成长链非编码RNA-BG的siRNA，通过脂质体介导的转染法转入Beas-2B细胞；采用实时定量PCR法检测干扰后Beas-2B细胞内BG的转录水平。实验分母细胞对照组、对照siRNA转染组和BG-siRNA转染组。采用克隆形成实验检测长链非编码RNA-BG对Beas-2B细胞受照射后增殖活性的影响；流式细胞术检测受照射后细胞周期的改变；通过免疫荧光染色检测电离辐射损伤后细胞内γ-H2AX焦点形成情况。采用Western blot法对DNA损伤相关蛋白（RAD50、磷酸化P53、KU70、KU80、MDM2、CDK2和RB蛋白）进行检测。结果 与对照siRNA转染组细胞相比，分别转染3组BG-siRNA 24h后，Beas-2B细胞内长链非编码RNA-BG的转录丰度均显著下降（t=8.32~15.29，P<0.05），后续实验采用干扰效率最高的2号siRNA进行。采用siRNA干扰长链非编码RNA-BG，经0、0.5、1、2、4和6GyX射线照射后2周，采用多靶单击模型拟合曲线发现，与Beas-2B母细胞对照组和对照siRNA转染组细胞相比，放射增敏比（SER）为0.80和0.82。转染BG-siRNA的Beas-2B细胞经4GyX射线照射24h后，细胞周期G2期比例由对照siRNA转染组的（37.37±0.63）%增至（64.19±1.01）%（t=30.65，P<0.05）；Beas-2B细胞内γ-H2AX焦点数目（59±3.49）/100个细胞较对照siRNA转染组（76±1.78）/100个细胞显著降低（t=13.72，P<0.05）；RAD50蛋白和磷酸化P53蛋白表达较对照siRNA转染组下降；KU70、KU80、MDM2、CDK2和RB蛋白较对照组表达增高。结论 靶向抑制长链非编码RNA-BG可能通过诱导细胞周期阻滞、促进DNA损伤修复，进而调控人正常支气管上皮细胞Beas-2B的放射敏感性。 |
| 英文摘要: |
| Objective To investigate the biological functions of lncRNA-BG on the radiosensitivity of normal human bronchial epithelial cell line Beas-2B.Methods Three lncRNA-BG siRNAs were designed, synthesized and transfected into Beas-2B cells via lipofectamine.The RNA transcription level of BG was detected by quantitative real time-PCR to confirm the siRNA transfection efficiency.The experiment was divided into control group, control siRNA transfected group, and BG transfected group.Cell survival was detected by clonogenic assay, and the cell cycle distribution was determined by flow cytometry assay.The γ-H2AX foci formation after irradiation was visualized via immunofluorescence.Western blot assay was performed to detect the protein expressions of RAD50,p-P53,KU70,KU80,MDM2,CDK2 and RB.Results BG-siRNA transfection significantly reduced the BG transcription level (t=8.32-15.29, P<0.05) and increased cell survival after irradiation at 0.5, 1, 2, 4 and 6 Gy.Analyzed with the multi-target model, the SERD0 of Beas-2B cells and control siRNA transfected cells were calculated to be 0.80 and 0.82, respectively.In addition, BG-siRNA transfection enhanced radiation-induced cell cycle arrest at G2 phase so that, after 4 Gy irradiation, the cells in G2 phase was increased from (37.37±0.63)% of control siRNA cells to (64.19±1.01)% (t=30.65, P<0.05).Meanwhile, the γ-H2AX foci of BG-siRNA transfected cells was decreased from 76±1.78 per 100 cells to 59±3.49 per 100 cells (t=13.72, P<0.05).The expressions of DNA damage related proteins including KU70, KU80, CDK2 and RB were increased, but the expressions of p-P53 and RAD50 were decreased.Conclusions LncRNA-BG could regulate the radiosensitivity of the normal human bronchial epithelial cells, probably through inducing cell cycle G2 phase arrest and promoting DNA damage repair after irradiation. |
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