APE1表达变化对人非小细胞肺癌细胞 A549增殖的影响

摘要

目的：探讨脱嘌呤/脱嘧啶核酸内切酶1（apurinic/ apyrimidinic endonuclease 1，APE1）对人非小细胞肺癌细胞 A549增殖能力的影响，为肺癌的个性化靶向治疗提供理论依据。方法：使用脂质体转染法将重组表达质粒 pSIREN - RetroQ - APE1- shRNA 和 pOZN - HA - APE1导入 A549细胞，免疫印迹检测 APE1表达情况。MTT、平板克隆实验、免疫印迹检测 APE1对非小细胞肺癌细胞增殖的影响，流式细胞仪检测细胞周期的变化。结果：重组载体 pSIREN - RetroQ - APE1- shRNA 转染细胞后，显著抑制 A549细胞中 APE1蛋白的表达，与对照组相比，增殖速率明显下降（P <0.05），平板克隆形成显著减少（P <0.05），并阻止细胞周期 G0/G1期向 S 期转化，降低 CDK2表达。而过表达 APE1可增加 A549细胞活力，促进细胞增殖，促进细胞周期G0/ G1期向 S 期转化。结论：APE1表达下调对非小细胞肺癌细胞 A549生长具有抑制作用，为临床抑制肿瘤生长提供了新的作用靶点。%Objective:To study the effect of lung cancer cells proliferation after silencing or over - expressing of APE1,providing a mineral strategy for individualized and target lung cancer therapy. Methods:The recombinant vec-tors with shRNA targeting APE1(pSIREN - RetroQ - APE1 - shRNA)and pOZN - HA - APE1(over expression of APE1)were transfected into A549 cells with the help of Lippofectamine 2000 to down or up regulate the expression of APE1. MTT assay and Colony Forming Assay were used to evaluate the proliferation and colony forming efficacy of A549 cells. Cell cycle was detected by flow cytometry(FCM). Western blot assay was performed to monitor the ex-pression level of protein APE1 and CDK2. Results:After transfection of the pSIREN - RetroQ - APE1 - shRNA targe-ting APE1,APE1 could be inhibited efficiently at protein level( P < 0. 05),cell proliferation was enormously de-creased compared to control groups(P < 0. 05),and G0 / G1 to S phase was inhibited and CDK2 expression was de-creased compared to control groups. However,after transfection of the pOZN - HA - APE1 targeting APE1(up - regu-lated APE1),vice versa. Conclusion:Stable silence of APE1 gene by shRNA could negatively regulate cell prolifera-tion in human non - small cell lung cancer cell line A549 cells. This experiment could provide a new insight into the mechanism of lung cancer and suggest a promising target of suppression cancer growth.