目的:探索葛根素抑制膀胱癌(bladder cancer,BLCA)T24及5637细胞系的作用机制.方法:利用Cell Counting Kit-8(CCK-8)检测法证实葛根素抑制BLCA T24及5637细胞的能力.通过串联质谱标签(Tandem Mass Tags,TMT)技术获得差异表达蛋白列表,并进行功能富集分析.通过生物信息分析筛选关键蛋白并对其进行表达分析、生存分析和上游转录因子预测.分子对接及Western blotting实验用于上游转录因子的验证.结果:CCK-8检测结果显示葛根素对T24和5637细胞系的IC50分别为218 μmol·L-1和198 µmol·L-1.功能富集分析显示差异表达蛋白主要富集在细胞周期和DNA复制通路.筛选出的关键蛋白为CDK1、CCNB1和PLK1.CHEA3网站预测关键蛋白上游转录因子为着丝粒蛋A(centromere protein A,CENPA.分子对接显示葛根素与 CENPA 的结合能为-6.3 kcal·mol-1(1 cal=4.184 0 J),Western blotting显示葛根素可显著降低CENPA的表达.结论:葛根素可能通过抑制CENPA的表达来影响蛋白CCNB1和PLK1,从而调控BLCA细胞的细胞周期或DNA复制以抑制其增殖.
TMT based protein quantitative analysis for the mechanism of puerarin inhibiting bladder cancer T24 cells proliferation
Objective:To investigate the mechanism of puerarin inhibiting bladder cancer(BLCA)T24 and 5637 cell lines.Methods:Cell Counting Kit-8(CCK-8)was used to confirm the proliferation inhibition of puerarin to BLCA T24 and 5637 cells.A list of differentially expressed proteins,on which the functional enrichment analysis was based,was obtained by Tandem Mass Tags(TMT)technology.Bioinformatics analysis was used to screen key proteins,on which the expression analysis,survival analysis,and upstream transcription factor prediction were per-formed.Molecular docking and Western blotting experiments were used to verify upstream transcription factor.Results:CCK-8 results showed that the IC50 of puerarin for T24 and 5637 cell lines were 218 μmol·L-1 and 198 µmol·L-1.Pathway enrichment analysis showed that the differentially expressed proteins were mainly en-riched in cell cycle and DNA replication pathway.The key proteins CDK1,CCNB1 and PLK1 were screened out,and CHE A3 predicted that the upstream transcription factor of the key protein was centromere protein A(CENPA).Molecular docking showed that the binding energy of puerarin and CENPA was-6.3 kcal·mol-1,and Western blotting showed that puerarin could significantly reduce the expression of CENPA.Conclusion:Puerarin may affect CCNB1 and PLK1 by inhibiting the expression of CENPA,thereby regulating the cell cycle or DNA replication of BLCA cells and inhibiting its proliferation.
puerarinbladder cancertandem mass tagscell cycleDNA replication
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