摘要
背景与目的肺癌耐药是肺癌患者死亡的主要原因,PPAR-γ可促进细胞凋亡,逆转肺癌耐药。本实验旨在探讨PPAR-γ表达下调对人肺癌A549顺铂耐受性和细胞凋亡的影响。方法构建siRNA沉默PPAR-γ的A549细胞系[A549/PPAR-γ(-)],应用MTT法检测顺铂对PPAR-γ沉默A549细胞增殖的影响,应用流式细胞术检测顺铂对PPAR-γ沉默A549细胞周期的影响,Western blot法检测磷酸化Akt(p-Akt)、caspase-3和bcl-2/bax的变化,最后以RT-PCR检测bcl-2的转录水平。结果成功构建出两个A549/PPAR-γ(-)细胞克隆,经RT-PCR和Western blot检测其PPAR-γ的水平明显下降。PPAR-γ沉默后,两个克隆A549细胞对顺铂的耐受性分别增加了1.29倍和1.60倍,肿瘤细胞的凋亡减少。Western blot检测显示Akt的磷酸化水平和bcl-2/bax水平升高,caspase-3表达降低,RT-PCR进一步显示bcl-2的转录水平升高。结论抑制A549中PPAR-γ的表达后,肿瘤细胞获得对顺铂药物更高的耐受性,其机制与升高Akt磷酸化水平和bcl-2表达水平,抑制细胞凋亡有关。PPAR-γ下调是临床肿瘤产生耐药性的可能机制之一。
Background and objective Drug resistance is the one of primary causes of death in patients with lung cancer,PPAR-γ could induce the apoptosis and reverse drug resistance.The aim of this study is to investigate the expression of PPAR-γ on cisplatin sensitivity and apoptosis response of human lung cancer cell line A549.Methods Reconstruction of PPAR-γ silencing A549 cells（A549/PPAR-γ（-）） by siRNA.MTT assay was employed to determine the effect of cisplatin on the proliferation of A549/PPAR-γ（-）,flow cytometry to determine the effect of cisplatin on the cell apoptosis,Western blot to determine the change of phosphorylation of Akt,caspase-3 and expression of bcl-2/bax.Finally,RT-PCR was employed to determine the transcriptional level of bcl-2.Results Two PPAR-γ silencing A549 cell clones were established successfully,and the expression of PPAR-γ was downregulated significantly as confirmed by RT-PCR and Western blot.After PPAR-γ silencing,the resistance of these two A549 clones to cisplatin was increased by 1.29-fold and 1.60-fold respectively.Flow cytometry showed that the apoptosis rate was decreased,and Western Blot showed that the phosphorylation of Akt and expression of bcl-2/bax were upregulated,caspase-3 was downregulated.Finally,RT-PCR showed that the transcriptional level of bcl-2 was upregulated as well.Conclusion Downregulation of PPAR-γ in A549 cells led to increase of cisplatin resistance.One of the mechanisms was upregulatin of phosphorylation of Akt and expression of bcl-2,which inhibited the apoptosis of cells.The downregulation of PPAR-γ is a possible mechanism that leads to the clinical drug resistance of cancer.
出处
《中国肺癌杂志》
CAS
北大核心
2013年第3期125-130,共6页
Chinese Journal of Lung Cancer
