染料木素对人鼻咽癌CNE1细胞生长的抑制作用及靶点预测  

Inhibitory Effect and Target Prediction of Genistein on the Growth of Human Nasopharyngeal Carcinoma CNE1 Cells

作　　者：何文栋 苏雯清 韦坤华[3,4] 奎玲 王硕[6] 龚小妹[6] 杨晓男 缪剑华 HE Wendong;SU Wenqing;WEI Kunhua;KUI Ling;WANG Shuo;GONG Xiaomei;YANG Xiaonan;MIAO Jianhua(Pharmacy College,Guangxi University of TCM,Nanning 530200,China;Pharmacy College,Guangxi Medical University,Nanning 530021,China;Guangxi Key Laboratory of Medicinal Resources Protection and Genetic Improvement,Guangxi Botanical Garden of Medicinal Plants,Nanning 530023,China;Guangxi Engineering Research Center of TCM Resource Intelligence Creation,Guangxi Botanical Garden of Medicinal Plants,Nanning 530023,China;Pharmacy College,Jiangsu University,Jiangsu Zhengjiang 212013,China;National Engineering Laboratory of Southwest Endangered Medicinal Resources Development,Guangxi Botanical Garden of Medicinal Plants,Nanning 530023,China)

机构地区：[1]广西中医药大学药学院,南宁530200 [2]广西医科大学药学院,南宁530021 [3]广西药用植物园广西药用资源保护与遗传改良重点实验室,南宁530023 [4]广西药用植物园广西壮族自治区中药资源智慧创制工程研究中心,南宁530023 [5]江苏大学药学院,江苏镇江212013 [6]广西药用植物园西南濒危药材资源开发国家工程实验室,南宁530023

出　　处：《中国药房》2021年第10期1196-1204,共9页China Pharmacy

基　　金：科技基础资源调查专项项目(No.2018FY100700);广西科技计划项目(No.桂科AD17129044);广西创新驱动发展专项资金项目(No.桂科AA18242040);2018级研究生教育创新计划立项课题(No.18210156)。

摘　　要：目的:研究染料木素对人鼻咽癌CNE1细胞生长的抑制作用并预测其可能的作用靶点。方法:采用CCK-8法检测0(空白对照)、12.5、25、50、100、150μmol/L染料木素分别作用24、48、72 h对CNE1细胞增殖的影响;分别采用流式细胞术检测0(空白对照)、15、30、60μmol/L染料木素作用24 h对CNE1细胞周期、凋亡的影响;采用划痕实验检测0(空白对照)、10、20、30μmol/L染料木素作用24 h对CNE1细胞迁移能力的影响。采用高通量测序法挖掘0(空白对照)、30μmol/L染料木素作用24 h后CNE1细胞中的差异基因,并通过实时荧光定量-聚合酶链式反应法(RT-qPCR)对上述细胞实验相关差异基因mRNA的表达情况进行验证。结果:与空白对照比较,12.5、25、50、100、150μmol/L染料木素对细胞的增殖均有显著的抑制作用(P<0.01),且呈浓度-时间-效应趋势;15、30μmol/L染料木素可使细胞周期阻滞于G0/G1期(P<0.05或P<0.01),30、60μmol/L染料木素可使细胞周期阻滞于G2/M期并显著促进其凋亡(P<0.05或P<0.01);10、20、30μmol/L染料木素可显著抑制细胞的迁移能力(P<0.01)。高通量测序共挖掘出2271个差异基因(padj<0.05),其中1154个基因上调、1117个基因下调;结合细胞实验结果共筛选出p53、p21、STC2、FGF2、CDK6、CYCLIN D、PI3K、AKT等8个潜在靶点差异基因。经RT-qPCR法验证,其中p53、p21、STC2、FGF2、CDK6、CYCLIN D、AKT等7个潜在靶点差异基因mRNA的表达均显著下调(P<0.05),与转录组测序结果基本一致。结论:染料木素能有效抑制人鼻咽癌CNE1细胞的生长;其抗鼻咽癌机制可能与抑制突变型p53基因的表达,恢复野生型P53蛋白功能以及抑制磷脂酰肌醇3激酶/蛋白激酶B通路的活性有关。OBJECTIVE:To study the inhibitory effects of genistein on the growth of human nasopharyngeal carcinoma.CNE1 cells and predict its potential target.METHODS:CCK-8 method was used to test the effects of 0(blank control),12.5,25,50,100,150μmol/L genistein on the proliferation of CNE1 cells after treated for 24,48,72 h.Flow cytometry was carried out to detect the effects of 0(blank control),15,30,60μmol/L genistein on the cell cycle and apoptosis of CNE1 cells after treated for 24 h.Scratch test was used to investigate the effects of 0(blank control),10,20,30μmol/L genistein on the migration ability of CNE1 cells after treated for 24 h.High throughput sequencing was conducted to discover the differential genes in CNE1 cells after treated with 0(blank control),30μmol/L genistein for 24 h.RT-qPCR assay was adopted to verify the mRNA expression of related differential genes in above trials.RESULTS:Compared with blank control,12.5,25,50,100,150μmol/L genistein showed significant inhibitory effect on the proliferation of CNE1 cells(P<0.01),in a concentration-time-effect manner;15,30μmol/L genistein could arrest CNE1 cell cycle at G0/G1 stage(P<0.05 or P<0.01);30,60μmol/L could arrest CNE1 cell cycle at G2/M stage and promoted cell apoptosis(P<0.05 or P<0.01).10,20,30μmol/L genistein could significantly inhibit the migration ability of CNE1 cells(padj<0.01).High throughput sequencing revealed a total of 2271 differentialgenes(P<0.05),1154 of which were up-regulated while 1117 of which were down-regulated;8 potential target genes,including p53,p21,STC2,FGF2,CDK6,CYCLIN D,PI3K,AKT,were screened by cell experiment.After validated by RT-qPCR assay,mRNA expression of p53,p21,STC2,FGF2,CDK6,CYCLIN D and AKT were significantly down-regulated(P<0.05),which consistent with the sequencing results.CONCLUSIONS:Genistein can effectively inhibit the growth of human nasopharyngeal carcinoma CNE1 cells,the mechanism of which may associated with inhibiting the expression of mutant gene p53,restoring the function of wild-type P53 pro

关 键 词：染料木素 人鼻咽癌CNE1细胞 高通量测序 突变型P53基因 磷脂酰肌醇3激酶/蛋白激酶B通路 

分 类 号：R285[医药卫生—中药学]