基于TMT蛋白质组学探讨华盖散对H1688和A549肺癌细胞增殖、凋亡影响的作用机制研究  

The Effect of Huagai Powder(华盖散) on the Proliferation and Apoptosis of H1688 and A549 Lung Cancer Cells and Its Mechanism:Based on TMT Protemoics Analysis

作　　者：霍炳杰[1] 宋彦茹 张洁[1] 李建波[1] 常靓 HUO Bingjie;SONG Yanru;ZHANG Jie;LI Jianbo;CHANG Liang(The Fourth Hospital of Hebei Medical University,Shijiazhuang,050011;Hebei University of Chinese Medicine)

机构地区：[1]河北医科大学第四医院,河北省石家庄市050011 [2]河北中医学院

出　　处：《中医杂志》2021年第16期1434-1440,共7页Journal of Traditional Chinese Medicine

基　　金：河北省中医药管理局科研计划(2019088)。

摘　　要：目的探讨华盖散的抗肿瘤活性及可能作用机制。方法选取对数生长状态良好的H1688和A549肺癌细胞以5×10^(3)/孔的密度接种于96孔板,细胞中分别加入125、250、500、1000μg/ml的华盖散(华盖散组),另设空白组,每组5个复孔。在37℃、5%CO_(2)条件下孵育处理肺癌细胞24、48、72h后,观察不同浓度华盖散作用不同时间对细胞增殖的影响及干预24h后对肺癌细胞凋亡的影响。采用TMT标记定量蛋白质技术联合液相色谱-串联质谱技术分析华盖散作用H1688细胞24h后肺癌细胞中的差异表达蛋白,并利用生物信息学分析筛选的差异蛋白,最后经Western blot方法验证差异蛋白结果。结果与空白组比较,华盖散组250、500、1000μg/ml浓度各时间均能够一定程度上降低H1688和A549细胞增殖率,提高细胞凋亡率,具有剂量依赖性(P<0.05)。蛋白质组学分析表明,华盖散干预能够引起112个蛋白的差异表达,其中上调蛋白58个,下调蛋白54个。筛选出与细胞增殖和凋亡相关的泛素样含PHD和环指域(UHRF1)、αB-晶状体蛋白(CRYAB)、生长抑制因子3(ING3)和PRKCZ凋亡WT1调节器(PAWR)蛋白进行Western blot验证,结果显示华盖散组ING3和PAWR蛋白表达下调,UHRF1和CRYAB蛋白表达上调(P<0.05)。结论华盖散可抑制H1688和A549肺癌细胞增殖,诱导凋亡,UHRF1、CRYAB、ING3和PAWR有可能为其治疗肺癌的生物标志。Objective To explore the anti-tumor effect of Huagai Powder(华盖散)and its possible mechanism.Methods H1688 and A549 cells in logarithmic growth and good condition were seeded in a 96-well plate at a density of 5×10^(3);different concentrations of Huagai Powder at 125,250,500 and 1000μg/ml were given while a blank group was set,with five compound holes in each group.After incubating the lung cancer cells at 37℃ and with 5%CO_(2) for 24,48,and 72 hours,the effects of different concentrations of Huagai Powder on cell proliferation were assessed,as well as and the effect on lung cancer cell apoptosis after 24 hours.Tandem Mass Tag(TMT)labeled quantitative protein technology combined with liquid chromatography tandem mass spectrometry was used to explore the differentially expressed proteins in the lung cancer cells after 24 hours' intervention of Huagai Powder on H1688 cells,and the differential proteins screened by bioinformatics analysis were verified by Western blotting.Results Compared to the blank group,Huagai Powder at different concentrations administered for different time could all inhibit the proliferation rate of H1688 and A549 cells and increase the apoptosis rate,and the effect was dose-dependent(P<0.05).Proteomics analysis showed that Huagai Powder could induce the differential expression of 112 proteins,including 58 up-regulated proteins and 54 down-regulated proteins.The UHRF1,CRYAB,ING3 and PAWR proteins related to cell proliferation and apoptosis were selected for Western blotting verification,and the results showed that the expression of ING3 and PAWR proteins was down-regulated while the expression of UHRF1 and CRYAB proteins was up-regulated in Huagai Powder group(P<0.05).Conclusion Huagai Powder can inhibit the proliferation and induce apoptosis of H1688 and A549 lung cancer cells,and UHRF1,CRYAB,ING3 and PAWR may be biomarkers for the treatment of lung cancer.

关 键 词：华盖散 肺癌 细胞增殖 细胞凋亡 蛋白质组学 

分 类 号：R285[医药卫生—中药学]