上调Numb通过调控铁死亡途径增加胃癌细胞化疗敏感性的机制研究

Mechanism of Upregulation of Numb to Increase Chemotherapy Sensitivity of Gastric Cancer Cells by Regulating Iron Death Pathway

目的探究在胃癌细胞中上调细胞命运决定子Numb对胃癌细胞化疗敏感性的影响及其作用机制。方法通过脂质体转染技术将pcDNA3.1空质粒、pcDNA3.1-Numb质粒分别转染至胃癌细胞系BGC-823中,实时荧光定量PCR(qRT-PCR)和蛋白质印迹检测转染后细胞中Numb表达变化,采用不同浓度的多柔比星(doxorubicin,ADM)处理转染后的BGC-823细胞,MTT法检测细胞增殖抑制率。将BGC-823细胞分为对照组、NC组、Numb组、Numb+铁死亡抑制剂Ferrostatin-1(Fer-1)组,MTT法检测各组细胞增殖抑制率,DCFH-DA荧光探针法检测各组细胞活性氧(reactive oxygen species,ROS)水平,试剂盒检测各组细胞谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)含量及超氧化物歧化酶(superoxide dismutase,SOD)活力,铁离子检测试剂盒测定各组细胞内铁离子浓度,RT-qPCR和蛋白质印迹法检测各组细胞中铁死亡相关途径因子P53、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)和溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)表达水平。结果与对照组、NC组比较,Numb组细胞中Numb mRNA和蛋白相对表达量均显著上调(P<0.05),在不同浓度ADM处理下的细胞增殖抑制率显著升高(P<0.05)。使用Fer-1干预并进行分组处理后,与对照组、NC组比较,Numb组细胞增殖抑制率显著升高(P<0.05),细胞中ROS水平、MDA含量显著增加(P<0.05),GSH含量显著减少(P<0.05),SOD活性显著降低(P<0.05),Fe^(2+)浓度显著升高(P<0.05),P53 mRNA和蛋白相对表达量显著上调(P<0.05),GPX4、SLC7A11的mRNA和蛋白相对表达量显著下调(P<0.05);与Numb组比较,Numb+Fer-1组BGC-823细胞增殖抑制率显著降低(P<0.05),细胞中ROS水平和MDA含量显著减少(P<0.05),GSH含量显著增加、SOD活性显著升高(P<0.05),同时细胞内Fe^(2+)浓度显著降低(P<0.05),P53 mRNA和蛋白相对表达量显著下调(P<0.05),而GPX4、SLC7A11的mRNA和蛋白相对表达量显著上调(P<0.05)。结论上调Numb能够提高胃癌细胞对ADM的化疗敏感性,该机制可能与其调控P53/SLC7A11/GPX4通路诱导铁死亡的作用有关。

Objective To investigate the effect of up-regulated cell fate determinant Numb on chemotherapy sensitivity of gastric cancer cells and its mechanism.Methods pcDNA3.1 plasmid vector and PCDNA3.1-Numb plasmid were transfected into gastric cancer cell line BGC-823 by liposome transfection technique.The transfected BGC-823 cells were treated with different concentrations of doxorubicin(ADM),and the cell proliferation inhibition rate was determined by MTT assay.BGC-823 cells were divided into 4 groups:control group,NC group,Numb group,and Numb+Ferrostatin-1(Fer-1)group,and the cell proliferation inhibition rate of each group was determined,and the level of reactive oxygen species(ROS)in each group was detected by DCFHDA fluorescent probe method.The levels of glutathione(GSH),malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)were measured by kits,and the concentration of iron ion in cells was determined by the iron ion detection kit.The expression levels of Numb,iron death related pathway factor P53,glutathione peroxidase 4(GPX4)and solute carrier family 7 member 11(SLC7A11)were detected by qRT-PCR and Western blotting.Results The mRNA and protein relative expression levels of Numb in the Numb group were significantly up-regulated(P<0.05),and the cell proliferation inhibition rate was significantly increased after ADM treatment(P<0.05)when compared with those in the control and NC groups.In comparison of those in the control group and NC group,the cell proliferation inhibition rate in the Numb group was significantly increased(P<0.05),the levels of ROS and MDA in cells were significantly increased(P<0.05),the level of GSH content and the activity of SOD was significantly decreased(P<0.05),the Fe^(2+) concentration was significantly increased(P<0.05),the expression level of P53 was significantly increased(P<0.05),and the expression levels of GPX4 and SLC7A11 were significantly decreased(P<0.05).After the intervention by Fer-1 treatment,the proliferation inhibition rate of BGC-823 cells in the Numb+Fer-1 group was significantly decreased(P<0.05),the levels of ROS and MDA were significantly decreased(P<0.05),the GSH level and SOD activity were significantly increased(P<0.05),the Fe^(2+) concentration was significantly decreased(P<0.05),the expression level of P53 were significantly decreased(P<0.05),and the expression levels of GPX4 and SLC7A11 were significantly up-regulated(P<0.05)when compared with those in the Numb group.Conclusion Up-regulation of Numb can enhance the sensitivity of gastric cancer cells to ADM,which may be related to the regulation of P53/SLC7A11/GPX4 pathway to induce ferroptosis.