BackgroundA strong association between Epstein-Barr virus (EBV) infection and nasopharyngeal carcinoma (NPC) has been widely recognized in recent decades. The aim of the present study was to investigate latent membrane protein 1 (LMP1) regulation of nasopharyngeal carcinoma (NPC) CNE-2 cell growth and then examine the effects of LMP1-knockout with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9on Epstein-Barr virus (EBV) infection and CNE-2 cell growth.MethodsHuman NPC CNE-2 cells were infected with the recombinant LMP1- and LMP2A-carrying lentivirus, and then examined for cell growth with the colony forming assay as well as for the activation of transcription of eukaryotic translation initiation factor 4E (eIF4E) with reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot. CRISPR/Cas9-mediated knockout of LMP1 or LMP2A was performed with a single-guide RNA (sgRNA) targeting sequences within LMP1 or LMP2A. The knockout effect and the EBV proliferation were examined with RT-qPCR, western blot and cell growth assay.ResultsLMP1 overexpression promoted CNE-2 cell growth, compared to LMP2A overexpression. Loss-of-function experiments confirmed that eukaryotic translation initiation factor 4E (eIF4E) upregulation mediated this effect. LMP1 knockout significantly inhibited EBV proliferation in CNE-2 cells and markedly inhibited LMP1-mediated promotion of cell growth. The knockout of either LMP1 or LMP2A blocked the eIF4E activation, which is induced either by the EBV infection or by the overexpression of LMP1 or LMP2A.ConclusionWe confirmed the LMP1-mediated promotion of NPC cell growth. Such promotion can be effectively blocked by CRISPR/Cas9-mediated LMP1 knockout. Precise LMP1 knockout might be a promising method for targeted inhibition of EBV infection and NPC cell growth.

中文翻译：

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CRISPR/Cas9 介导的 LMP1 敲除抑制 Epstein-Barr 病毒感染和鼻咽癌细胞生长

背景近几十年来，爱泼斯坦-巴尔病毒 (EBV) 感染与鼻咽癌 (NPC) 之间的密切关联已得到广泛认可。本研究的目的是研究潜伏膜蛋白 1 (LMP1) 对鼻咽癌 (NPC) CNE-2 细胞生长的调节，然后检查 LMP1 敲除与 CRISPR（成簇规则间隔短回文重复）/Cas9 对 Epstein 的影响-巴尔病毒 (EBV) 感染和 CNE-2 细胞生长。方法用携带 LMP1 和 LMP2A 的重组慢病毒感染人 NPC CNE-2 细胞，然后用集落形成测定法检查细胞生长情况以及活化情况用逆转录酶定量聚合酶链反应 (RT-qPCR) 和蛋白质印迹法对真核翻译起始因子 4E (eIF4E) 进行转录。CRISPR/Cas9 介导的 LMP1 或 LMP2A 敲除是使用 LMP1 或 LMP2A 内的单向导 RNA (sgRNA) 靶向序列进行的。用RT-qPCR、蛋白质印迹和细胞生长测定法检测敲除效应和EBV增殖。结果与LMP2A过表达相比，LMP1过表达促进CNE-2细胞生长。功能丧失实验证实真核翻译起始因子 4E (eIF4E) 上调介导了这种效应。LMP1 敲除显着抑制了 CNE-2 细胞中的 EBV 增殖，并显着抑制了 LMP1 介导的细胞生长促进作用。LMP1或LMP2A的敲除阻断了eIF4E的激活，这是由EBV感染或LMP1或LMP2A的过表达诱导的。结论我们证实了LMP1介导的NPC细胞生长促进作用。CRISPR/Cas9 介导的 LMP1 敲除可以有效地阻止这种促进作用。精确的 LMP1 敲除可能是靶向抑制 EBV 感染和 NPC 细胞生长的有前途的方法。