NEDD8结合酶UBE2F调控肺腺癌转移的分子机制研究

Molecular mechanism of NEDD8-conjugating enzyme UBE2F regulating lung adenocarcinoma metastasis

目的:探究NEDD8结合酶UBE2F对肺腺癌(lung adenocarcinoma,LUAD)转移的影响。方法:利用TIMER2.0、UALCAN、HPA等数据库分析UBE2F在肺腺癌中的表达情况;利用Kaplan-MeierPlotter数据库分析UBE2F表达与肺腺癌生存率的关系;运用CRISPR/Cas9技术构建UBE2F敲除的肺腺癌细胞系,并构建UBE2F敲除的裸鼠肺腺癌转移瘤模型,验证UBE2F敲除对肺腺癌转移的影响;通过细胞划痕实验以及Transwell迁移和侵袭实验检测UBE2F敲除对肺腺癌细胞侵袭、迁移能力的影响;Westernblot法和RealtimePCR法检测下调UBE2F表达对上皮-间充质转化(epithelial-mesenchymal transformation,EMT)转录因子Snail表达的影响。结果:多个数据库分析表明,UBE2F在肺腺癌中高表达,且高表达患者比低表达患者具有更好的预后;体内实验表明,与对照组相比,UBE2F敲除后裸鼠肺表面转移的小结节数目显著增多(P<0.05);细胞划痕实验以及Transwell实验表明,UBE2F敲除增强肺腺癌细胞的迁移和侵袭能力,差异具有统计学意义(P<0.05);Westernblot和Re-altimePCR结果显示,UBE2F敲除升高EMT转录因子Snail蛋白水平和mRNA水平。结论:敲除UBE2F诱导Snail积聚,进而导致肺腺癌细胞的侵袭和转移。

AIM:To study the effect of NEDD8-conjugating enzyme UBE2F on lung adenocarcino-ma metastasis.METHODS:The expression of UBE2F in lung adenocarcinoma was analyzed using TIMER2.0,UALCAN and HPA databases.Kaplan-Meier Plotter database was used to analyze the rela-tionship between UBE2F expression and survival rate of lung adenocarcinoma.A UBE2F-knockout lung adenocarcinoma cell line was constructed using CRISPR/Cas9 technology,and a UBE2F-knockout lung adenocarcinoma metastasis model was con-structed in nude mice to verify the effect of UBE2F knockout on lung adenocarcinoma metastasis.The effects of UBE2F knockout on invasion and migra-tion of lung adenocarcinoma cells were examined by cell scratch assay and Transwell invasion and mi-gration assays.The effect of down-regulated UBE2F expression on snail expression,a key marker of epi-thelial-mesenchymal transition(EMT),was detect-ed by Western blot and Real time PCR.RESULTS:Multiple database analysis showed that UBE2F was highly expressed in lung cancer,and Kaplan-Meier Plotter analysis showed that high expression of UBE2F in lung adenocarcinoma had better progno-sis than low expression.In vivo experiments showed that compared with control group,the number of nodules metastasized on the lung sur-face of nude mice after UBE2F knockout was signifi-cantly increased(P<0.05).Cell scratch assay and Transwell assay showed that UBE2F enhanced the migration and invasion ability of lung cancer cells after knockout,and the difference were statistically significant(P<0.05).Western blot and Real time PCR results indicated that the level of EMT tran-scription factor snail protein and mRNA increased after UBE2F knockout.CONCLUSION:In lung ade-nocarcinoma cells,UBE2F down-regulation leads to Snail accumulation and promotes invasion and me-tastasis of lung adenocarcinoma cells.