冬凌草甲素联合免疫因子TRAF4对非小细胞肺癌细胞自噬和凋亡的影响及机制研究

Study on the effect of oridonin combined with immune factor TRAF4 on autophagy and apoptosis of non-small cell lung cancer cells and its mechanism

关键词:  冬凌草甲素  TRAF4  非小细胞肺癌  自噬  凋亡

Key words:  oridonin  TRAF4  non-small cell lung cancer  autophagy  apoptosis

摘要:

[目的] 探讨冬凌草甲素联合免疫因子肿瘤坏死因子受体相关因子4（TRAF4）对非小细胞肺癌细胞自噬和凋亡的影响及机制研究。[方法] 选择不同浓度的冬凌草甲素处理A549细胞，然后采用噻唑蓝（MTT）法检测细胞的活力。体外培养A549细胞，将细胞分为对照组、冬凌草甲素组、si-NC组（转染si-NC）、si-TRAF4组（转染si-TRAF4）、冬凌草甲素+pcDNA组（转染pcDNA后，选择冬凌草甲素浓度为20 μmol/L处理）、冬凌草甲素+TRAF4组（转染TRAF4后，选择冬凌草甲素浓度为20 μmol/L处理）。采用流式细胞术检测细胞凋亡；蛋白免疫印迹法（Western Blot）检测B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、微管相关蛋白1的轻链3（LC3）、Ⅱ/LC3Ⅰ、Beclin 1、自噬相关蛋白7（ATG7）和TRAF4蛋白表达；实时荧光定量聚合酶链反应（qRT-PCR）检测TRAF4 mRNA表达量。[结果] 不同浓度冬凌草甲素明显抑制细胞活力。冬凌草甲素组细胞凋亡率、Bax、Beclin 1、LC3Ⅱ/LC3Ⅰ、ATG7蛋白表达明显高于对照组；TRAF4 mRNA和蛋白表达、Bcl-2蛋白表达明显低于对照组。si-TRAF4组TRAF4 mRNA和蛋白表达、Bcl-2蛋白表达明显低于si-NC组；细胞凋亡率、Bax、Beclin 1、LC3Ⅱ/LC3Ⅰ和ATG7蛋白表达明显高于si-NC组。冬凌草甲素+TRAF4组TRAF4 mRNA和蛋白表达、Bcl-2蛋白表达明显高于冬凌草甲素+pcDNA组；细胞凋亡率、Bax、Beclin 1、LC3Ⅱ/LC3Ⅰ和ATG7蛋白表达明显低于冬凌草甲素+pcDNA组。[结论] 冬凌草甲素通过下调TRAF4的表达抑制非小细胞肺癌细胞的凋亡和自噬。

Abstract:

[Objective] To investigate the effect of oridonin combined with immune factor TRAF4 on autophagy and apoptosis of non-small cell lung cancer cells and its mechanism. [Methods] Different concentrations of oridonin were selected to treat A549 cells, and then MTT method was used to detect cell viability. Culture A549 cells in vitro and divide the cells into control group, oridonin group, si-NC group(transfected with si-NC), si-TRAF4 group(transfected with si-TRAF4), oridonin+pcDNA group(after transfection of pcDNA, select the oridonin concentration of 20 μmol/L for treatment), oridonin+TRAF4 group(after transfection of TRAF4, select the oridonin concentration for treatment of 20 μmol/L). Flow cytometry was used to detect cell apoptosis;Western blot were used to detect the protein expression of Bax, Bcl-2, LC3Ⅱ/LC3Ⅰ, Beclin 1, ATG7 and TRAF4;qRT-PCR detection of TRAF4 mRNA expression. [Results] Cell viability was suppressed by different concentrations of oridonin. The cell apoptosis rate, Bax, Beclin 1, LC3Ⅱ/LC3Ⅰ, and ATG7 protein expression in oridonin group were significantly higher than those of the control group;TRAF4 mRNA and protein expression, and Bcl-2 protein expression were significantly lower than those of the control group. The expression of TRAF4 mRNA and protein, and the expression of Bcl-2 protein in the si-TRAF4 group were significantly lower than those in the si-NC group;the apoptosis rate, Bax, Beclin 1, LC3Ⅱ/LC3Ⅰ and ATG7 protein expression were significantly higher than those in the si-NC group. The expression of TRAF4 mRNA and protein and Bcl-2 protein in the oridonin + TRAF4 group was significantly higher than that of the oridonin+pcDNA group;the apoptotic rate, Bax, Beclin 1, LC3Ⅱ/LC3Ⅰ and ATG7 protein expression were significantly lower in the oridonin+pcDNA group. [Conclusion] Oridonin inhibits the apoptosis and autophagy of non-small cell lung cancer cells by down-regulating the expression of TRAF4.