siRNA 靶向沉默 Lipocalin-2基因增加人肺癌 A549细胞对顺铂的敏感性

摘要

Objective To observe the effects of Lipocalin-2 silencing gene on the sensitivity of human lung cancer A549 cells to cisplatin through silencing the expression of Lipocalin-2 gene by means of small interfering RNA( siRNA). Methods The expression levels of Lipocalin-2 protein in lung cancer tissues and 3 kinds of human lung cancer cell lines including A549,NCI-H661,NCI-H446 were detected by immunohistochemistry( SP)and Western Blot,respectively. Specific Lipocalin-2 siRNA was established and transfected into A549 cells via lentivirus,then the transfection efficiency was detected by Real-time PCR and Western Blot. Cell survival rate and inhibitory concentration 50(IC50)were measured by MTT assay. The cell apoptosis rates after treatment by cisplatin were detected by flow cytometry. Results The overexpressions of Lipocalin-2 protein were found in lung cancer tissue and the three kinds of cancer cell lines( P < 0. 05). The expression levels of Lipocalin-2 mRNA and protein were significantly decreased in A549 cells after transfection by siRNA-Lipocalin-2,at the same time,the cell survival rate was obviously decreased after induction by cisplatin,however,cell apoptosis rate was significantly increased( P < 0. 05). Conclusion The specific siRNA can effectively inhibit the expression levels of Lipocalin-2 mRNA and protein in A549 cell and can enhance the sensitivity of A549 cells to cisplatin.%目的：利用针对 Lipocalin-2的小干扰 RNA（siRNA）沉默人肺癌 A549细胞中 Lipocalin-2基因表达，观察其对顺铂化疗敏感性的影响。方法分别采用免疫组织化学 SP 法和 Western blot 检测 Lipocalin-2在肺癌组织和3种肺癌细胞株（A549、NCI-H661、NCI-H446）中的表达情况。设计并构建靶向 Lipocalin-2的 siRNA 转染 A549细胞，采用Real-time PCR 和 Western blot 检测转染效率。MTT 法检测顺铂处理后细胞存活率及半数抑制浓度（IC50）。流式细胞术检测顺铂处理后细胞凋亡率。结果 Lipocalin-2蛋白在肺癌组织和肺癌 A549、NCI-H661、NCI-H446细胞中均异常高表达（ P ＜0.05）。siRNA-Lipocalin-2转染 A549细胞能在 mRNA 和蛋白水平显著抑制 Lipocalin-2表达，同时顺铂诱导的细胞存活率显著降低，凋亡率显著增高（ P ＜0.05）。结论利用特异性 siRNA 能有效抑制人肺癌 A549细胞中Lipocalin-2 mRNA 和蛋白表达，增强细胞对顺铂的敏感性。