PKM2基因对CD44^+肺癌干细胞增殖及耐药性的影响

Effect of pkm2 Gene on Proliferation and Drug Resistance in CD44^+ Lung Cancer Stem Cells

目的观察转染后CD44+肺癌干细胞内PKM2基因的表达变化及其对CD44+肺癌干细胞增殖、耐药性的影响。方法将A549肺癌细胞株继代培养后,采取免疫磁珠细胞分选法,通过Auto MACS仪器,分选出CD44+细胞。实验分为实验组和对照组,将人工合成的针对PKM2基因的siRNA片段通过Lipofectamine 2000转染细胞,分别采用实时荧光定量聚合酶链反应(real-time quantitative PCR,qRT-PCR)和Western blot法检测各组细胞中PKM2的mRNA和蛋白质的表达量,细胞免疫组化染色分析CD44及PKM2表达,并采用MTT法检测各组细胞的增殖活力。药性敏感性试验:在培养基内添加不同浓度化疗药物DDP,并通过MTT assay测试细胞的生长及增殖速度。结果 CD44+肺癌干细胞转染PKM2 siRNA后,CD44+肺癌干细胞中PKM2 mRNA及蛋白表达减弱(P<0.05),细胞增殖能力减弱,抗肿瘤药物耐药性降低。结论 PKM2对CD44+肺癌干细胞的增殖起促进作用,同时具有增强抗肿瘤药物耐药性的作用。

Objective To investigate the expression of PKM2 and its effect on the proliferation and drug resistance in CD44+lung cancer stem cells. Methods Lung cancer A549 cells were separated into CD44+cells and CD44+cells by the immunomagnetic beads method through Auto MACS instrument,and then the PKM2 siRNA was transfected into CD44+lung cancer stem cells by Lipofectamine 2000. The experimental group and the control group were included in the study. qRT-PCR and western blot were used to measure the PKM2 expression at mRNA and protein levels,respectively. The cell proliferation was assessed by MTT assay. Drug sensitivity test: different chemotherapy drugs solubility of DDP were added into the culture medium,and then the cell proliferation was assessed by MTT assay. Results PKM2 siRNA was transfected into CD44+lung cancer stem cells,the expression of PKM2 mRNA and protein in CD44+cells decreased significantly( P < 0. 05),cell proliferation ability decreased,and anti-tumor drug resistance was reduced in CD44+cells. Conclusion PKM2 plays an important role in promoting the proliferation of CD44+lung cancer stem cells and enhancing the anti-tumor drug resistance.