葛根素调控CENPA抑制膀胱癌细胞增殖的机制研究  

TMT based protein quantitative analysis for the mechanism of puerarin inhibiting bladder cancer T24 cells proliferation

作　　者：许浩 潘登 马雨阳 徐鹏[2] 王靖凯 王海跞 庞昆[4] 韩从辉 XU Hao;PAN Deng;MA Yuyang;XU Peng;WANG Jingkai;WANG Hailuo;PANG Kun;HAN Conghui(Graduate School,Bengbu Medical University,Bengbu 233000,China;Graduate School,Jiangsu University,Zhenjiang 212000,China;Graduate School,Xuzhou Medical University,Xuzhou 221000,China;Department of Urology,Xuzhou Central Hospital/Xuzhou Clinical School of Xuzhou Medical University/Xuzhou Central Hospital Affiliated to Nanjing University of Chinese Medicine,Xuzhou 221000,China)

机构地区：[1]蚌埠医科大学研究生院,安徽蚌埠233000 [2]江苏大学研究生院,江苏镇江212000 [3]徐州医科大学研究生院,江苏徐州221000 [4]徐州市中心医院/徐州医科大学徐州临床医学院/南京中医药大学附属徐州中心医院泌尿外科,江苏徐州221000

出　　处：《东南大学学报（医学版）》2024年第4期487-497,共11页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(82004110);中国博士后基金面上项目(2022M722674);徐州市医学后备人才计划项目(XWRCHT20220009);蚌埠医学院研究生科研创新计划(Byycx22129)。

摘　　要：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IC 50分别为218μmol·L^(-1)和198μmol·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=4.1840 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。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 performed.Molecular docking and Western blotting experiments were used to verify upstream transcription factor.Results:CCK-8 results showed that the IC 50 of puerarin for T24 and 5637 cell lines were 218μmol·L^(-1)and 198μmol·L^(-1).Pathway enrichment analysis showed that the differentially expressed proteins were mainly enriched in cell cycle and DNA replication pathway.The key proteins CDK1,CCNB1 and PLK1 were screened out,and CHEA3 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.

关 键 词：葛根素 膀胱癌 串联质谱标签 细胞周期 DNA复制 

分 类 号：R285.5[医药卫生—中药学] R737.14[医药卫生—中医学]