缺氧/放射活化Epo/e9增强子调控Layilin siRNA表达以抑制人肺腺癌A549细胞侵袭的研究

Inhibitory effects of layilin siRNA expression regulated by Epo/e9 enhancer activated through hypoxia/radiation on invasion behavior of human lung carcinoma cell A549 induced by hyaluronan in vitro

背景与目的:Layilin是一种新发现与肺腺癌侵袭转移有密切联系的特异性透明质酸受体。本研究旨在观察缺氧/放射条件下活化缺氧/放射双敏感性增强子(Epo/e9),调控针对layilin的小干扰RNA(small interfering RNA,siRNA)表达,探讨其对人肺腺癌A549细胞受透明质酸诱导侵袭的影响。方法:构建携带Epo/e9增强子和U6启动子且表达针对layilin的siRNA的RNA干扰载体(Epoo/e9-siLay plasmid)和阴性对照RNA干扰载体(Epo/e9-siCtrl plasmid),分别转染A549细胞,新霉素抗性筛选得到layilin表达受抑制的A549_(Epo/e9-siLay)细胞和layilin表达未受影响的A549_(Epo/e9-siCtrl)细胞。针对非转染A549(A549_(untransfected))、A549_(Epo/e9siLay)、A549_(Epo/e9-siCtrl)三组细胞,在缺氧/放射处理下,分别采用RT-PCR和和Western blot检测layilin mRNA和和蛋白表达,用Transwell模型检测细胞侵袭能力。结果:与A549_(untransfected)组相比,A549_(Epo/e9-siLay)组layilin表达显著减弱(P<0.01),Transwell穿膜细胞数显著减少(P<0.01);A549_(Epo/e9-siCtrl)组layilin表达、Transwell穿膜细胞数皆和A549_(untransfected)组无显著差异(P>0.05)。缺氧/放射处理能进一步增加和RNA干扰的上述效应(P<0.01)。结论:在缺氧/放射条件下,Epo/e9增强子调控layilin siRNA表达能明显抑制A549细胞侵袭行为。

Background and purpose：Layilin is a novel and special hyaluronan（HA） receptor that closely linked with lung cancer invasion and metastasis.The objective of this study is to investigate the effect of hypoxia/ radiation dual-sensitive enhancer（Epo/e9） regulating small interfering RNA（siRNA） for layilin expression on invasion of human lung adenocarcinoma A549 cells induced by HA in vitro.Methods：RNA interference plasmid including Epo/e9 enhancer and U6 promoter and expressing siRNA targeting layilin（Epo/e9-siLay plasmid） or expressing siRNA not matching any known human coding mRNA（Epo/e9-siCtrl plasmid） was designed,constructed,and lipotransfected into A549 cells line.A549^（Epole9-siLay） cells expressing suppressed layilin or A549^（Epole9-siCtrl） cells expressing uninfluenced layilin were selected by neomycin resistance.In A549^（undransfected＇） A549^（Epole9-siLay） and A549^（Epole9-siCtrl） groups,layilin mRNA and protein expression were detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot,and the invasion ability was examined by Transwell model with or without administration of hypoxia/radiation.Results： Compared with that in A549^（untransfected） group,layilin mRNA and protein expression,and the number of penetrating cells（NPC） were decreased significantly in A549^（Epole9-siLay） group（P0.01）,but there were no significant changes in A549^（Epole9-siCtrl） group（P0.05）.Administration of hypoxia/radiation reinforced the above effects of siRNA targeting layilin. Conclusion：Epole9 enhancer regulating siRNA for layilin expression can inhibit efficiently the invasive ability of human lung carcinoma cell line A549 induced by HA in vitro with hypoxia/radiation administration.