萤光素酶报告基因细胞模型在筛选靶向调控RAC1的大黄酸衍生物中的应用

Construction of luciferase reporter gene cell model and its application in screening rhein derivatives targeting RAC1

目的 构建含RAC1启动子萤光素酶报告基因CNE1-RAC1-Luc2稳转细胞模型,探讨其在转录水平筛选靶向调控RAC1抗肿瘤活性的大黄酸衍生物中的应用。方法 利用RAC1启动子序列,设计合成萤光素酶报告基因-慢病毒重组载体,感染鼻咽癌CNE1细胞,获得稳定表达萤光素酶的细胞;采用萤光素酶报告基因检测试剂盒,检测RAC1激活剂PMA和抑制剂NSC23766刺激该细胞后的萤光素酶发光值,并观察系列大黄酸衍生物对RAC1启动子萤光素酶活性的影响,Western blot验证细胞RAC1蛋白表达的变化。结果 双酶切实验显示,含RAC1启动子萤光素酶报告基因的慢病毒表达载体构建成功;经嘌呤霉素筛选,获稳定表达萤光素酶的CNE1-RAC1-Luc2细胞,转染效率达90%以上;RAC1萤光素酶报告基因检测系统对RAC1激活剂和抑制剂反应灵敏,萤光素酶活性与Western blot实验中RAC1蛋白表达结果一致;系列大黄酸衍生物对CNE1-RAC1-Luc2细胞萤光素酶活性的调控作用与Western blot结果基本一致。结论 成功构建了含RAC1启动子的萤光素酶报告系统的细胞模型,为高通量进行靶向RAC1药物的筛选提供一个实用的平台。

Aim To establish luciferase reporter gene expression cell models of CNE1-RAC1-Luc2 based on the target of RAC1 promoter, and explore the application of screening anti-tumor active of rhein derivatives targeted regulating RAC1 at transcriptional level.Methods The lentiviral carrying luciferase reporter vector was designed and synthesized using RAC1 promoter sequence, and CNE1 cells were infected with recombinant plasmid lentiviral to obtain cell lines that stably expressed firefly luciferase.Luciferase reporter assay was used to detect the luciferase luminescence value after stimulating with RAC1 activator PMA and inhibitor NSC23766 that targeted regulating the RAC1 pro- moter activities in cells, and RAC1 expression was verified by Western blot.The effect of series of rhein derivatives on the luciferase activity of RAC1 promoter was observed, and RAC1expression was determined by Western blot.Results The identification result of double enzyme digestion showed that a lentiviral expression vector carrying luciferase reporter vector recombined with RAC1 promoter was successfully constructed.Lentivirus-infected CNE1 cells were screened by puromycin, the CNE1-RAC1-Luc2 cells stably expressing firefly luciferase were obtained, and the transfection efficiency was over 90%.The RAC1 luciferase reporter assay system was sensitive to PMA and NSC23766 and consistent with the result of RAC1 protein expression by Western blot.The regulation of series of rhein derivatives to RAC1 luciferase activity of CNE1-RAC1-Luc2 cells was consistent with the results of Western blot.Conclusions The cell model of luciferase reporting system containing RAC1 promoter can be successfully constructed, which provides a practical platform for high throughput screening of RAC1-targeted drugs.