过表达EphA2促进潜伏期稳定感染EBV肺癌细胞株的建立
Overexpression of EphA2 Promotes the Establishment of Lung Cancer Cell Line With Stable Latent EB Virus Infection
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摘要:目的 建立一株稳定潜伏期感染EB病毒（Epstein-Barr virus，EBV）的肺癌上皮细胞，并探究EBV的感染对肺癌细胞表型的影响。方法 通过慢病毒在肺癌细胞系中过表达EphA2，将该细胞与携带EBV-eGFP的Akata B细胞直接接触式共培养，用G418富集成功感染EBV的上皮细胞，通过EBNA1免疫荧光验证EBV的感染。流式分选出携带绿色荧光的上皮细胞培养10代以上，对稳定携带绿色荧光的A549-EphA2-EBV细胞进行克隆形成、划痕实验、Transwell迁移和侵袭等表型实验，观察EBV感染对肺癌细胞表型的影响。结果 A549亲本细胞与Akata-EBV-eGFP直接接触式共培养难以在短时间内实现EBV的感染，EphA2过表达显著促进了EBV在肺癌细胞中的感染效率，感染效率为0.53%。将流式分选出的细胞经过10代以上培养仍观察到绿色荧光蛋白的表达，EBER原位杂交说明该细胞中仍存在EBV的感染，证实该细胞株中EBV为稳定潜伏期感染。结论 通过过表达EphA2建立了一株稳定潜伏期感染EBV的肺癌上皮细胞，为EBV相关肺癌发生发展机制探索提供了材料基础。该潜伏期EBV感染的肺癌上皮细胞增殖迁移等恶性表型受到抑制，其相关分子机制有待深入研究。Abstract:Objective To establish an Epstein-Barr virus (EBV) infectious lung cancer cells mediated by EphA2 overexpression.Methods A lentiviral system was employed to establish a stable cell line with ectopic overexpression of EphA2 in lung cancer cell line. These engineered cells were subsequently co-cultured with Akata B cells which carry EBV-eGFP. Following co-culture, successfully infected epithelial cells were selected using G418 and validated by immunofluorescence of EBNA1. Phenotypic assays, including colony formation, wound healing, transwell migration, and invasion assays, were then performed on the established A549-EphA2-EBV cell line to assess the effects of EBV infection.Results It is difficult to achieve EBV infection in a short period of time through direct contact co-culture of A549 parental cells with Akata-EBV-eGFP. EphA2 overexpression significantly enhanced the infection efficiency of EBV in lung cancer cells, achieving an infection efficiency of 0.53%. Green fluorescent protein expression was observed after more than ten passages of culture, and EBER in situ hybridization confirmed the persistence of EBV infection, demonstrating that the cell line harbors EBV in a stable latent state.Conclusions We established a lung cancer epithelial cell line with stable latent EBV infection by EphA2 overexpressing, providing essential material for investigating the mechanisms underlying pulmonary lymphoepithelioma carcinoma. The malignant phenotypes, including proliferation and migration, of these latently EBV-infected lung cancer epithelial cells were suppressed, and the underlying molecular mechanisms warrant further investigation.
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