高丽槐素对鼻咽癌CNE1细胞增殖、迁移、凋亡的影响及机制  

Influence and mechanism of maackiain on proliferation, migration, and apoptosis of nasopharyngeal carcinoma CNE1 cells

作　　者：苏雯清 杨晓男 何文栋 韦坤华 王硕 龚小妹 奎玲 缪剑华[1,2,3,4,5] Su Wenqing;Yang Xiaonan;He Wendong;Wei Kunhua;Wang Shuo;Gong Xiaomei;Kui ling;Miao Jianhua(Pharmaceutical College,Guangxi Medical University,Nanning 530021,China;Guangxi University of Chinese Medicine,Nanning 530200,China;National Engineering Laboratory of Southwest Endangered Medicinal Resources Development,Nanning 530023,China;Guangxi Key Laboratory of Medicinal Resources and Genetic Improvement,Nanning 530023,China;Guangxi Zhuang Autonomous Region Traditional Chinese Medicine Resources intelligent Creation Engineering Research Center,Nanning 530023,China;School of Pharmacy,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]广西医科大学药学院,南宁530021 [2]广西中医药大学,南宁530200 [3]广西药用资源保护与遗传改良重点实验室,南宁530023 [4]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]西南濒危药材资源开发国家工程实验室,南宁530023 [6]江苏大学药学院,镇江2120136

出　　处：《广西医科大学学报》2021年第2期258-264,共7页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.82060689);广西创新驱动发展专项资金项目“药用植物大数据中心建设”基金资助项目(No.AA18242040);科技基础资源调查专项项目“‘一带一路’国家传统草药品种本底整理及数据库建设”基金资助项目(No.2018FY100700);广西科技计划项目“珍稀濒危药用资源保护与可持续利用工作站”基金资助项目(No.AD17129044)。

摘　　要：目的:探讨高丽槐素对鼻咽癌(NPC)细胞CNE1增殖、单克隆、迁移和凋亡的影响及潜在的作用靶点。方法:将培养的CNE1细胞随机分为空白对照组和高丽槐素给药组。采用CCK8法检测不同浓度的高丽槐素作用24 h、48 h、72 h后的增殖抑制率,单克隆试验检测各组细胞的克隆形成能力,细胞划痕试验检测各组细胞迁移能力,流式细胞术试验检测细胞的凋亡情况,利用高通量测序技术,分析CNE1细胞转录组的变化,得到差异表达基因,筛选潜在基因靶点,采用反转录实时荧光定量PCR(RT-qPCR)进行验证。结果:CCK8结果显示,高丽槐素能显著抑制CNE1细胞的增殖,且具有时间和浓度依赖性(P<0.05);与对照组比较,高丽槐素能抑制CNE1细胞的克隆能力和迁移能力(P<0.05),且CNE1细胞克隆形成能力与迁移能力随着高丽槐素浓度的增加而减弱;Annexin V-FITC/PI染色结果显示,高丽槐素可使CNE1细胞凋亡率升高(P<0.05);高通量测序结果显示,共有4 232个差异表达基因,表达程度显著升高的基因数有1 060个,表达程度显著降低的基因数有3 172个,高丽槐素可使抑癌基因(P53)、细胞周期依赖激酶抑制剂(P21)、成纤维细胞生长因子(Fgf2)、磷脂酰肌醇3-激酶(Pi3k)、丝裂原活化蛋白激酶8(Mapk8)等基因下调(P<0.05);RT-qPCR显示高丽槐素能下调P53、P21、Fgf2、Pi3k、Mapk8 mRNA表达水平(P<0.05),检测结果显示与测序结果相一致。结论:高丽槐素可抑制CNE1细胞的增殖、克隆与迁移能力并促进细胞凋亡,其作用机制可能与P53、P21、Fgf2、pi3k、Mapk8等多组基因的转录变化有关。Objective: To investigate the effects of maackiain on proliferation, monoclonal, migration and apoptosis of nasopharyngeal carcinoma(NPC) cells and its potential targets. Methods: Cultured CNE1 cells were divided into two groups: control group and maackiain treated group. CCK8 test was used to detect the proliferation inhibition rate of different concentrations of maackiain after 24 h, 48 h and 72 h. Monoclonal test was used to detect the clonogenesis ability of each group. The cell migration ability of each group was evaluated by cell scratch test. Flow cytometry was used to detect the apoptosis of the cells. High-throughput sequencing was used to analyze the transcriptome changes of CNE1 cells, and the differentially expressed genes were obtained. Potential gene targets were screened and validated by RT-q PCR. Results: CCK8 showed that maackiain could significantly inhibit the proliferation of CNE1 cells in a time-concentration-dependent manner(P<0.05).Compared with the control group, the clonal capacity(P<0.05) and migration capacity(P<0.05) of CNE1 cells were inhibited by maackiain, and the clonal formation capacity and migration capacity of CNE1 cells were decreased with the increase of maackiain concentration. Annexin V-FITC/PI staining showed that maackiain increased apoptosis rate of CNE1 cells(P<0.05). High-throughput sequencing showed that there were 4,232 expressed genes, among which 1,060 genes were overexpressed and 3,172 genes were underexpressed. RT-q PCR showed that maackiain could down-regulate the m RNA levels of tumor suppressor gene P53, Cyclin-dependent kinases inhibitor P21, fibroblast growth factor 2(Fgf2), phosphatidylinositol 3-kinase(Pi3 k) and recombinant mitogen activated protein kinase 8(Mapk8)(P<0.05). These test results were the same as the sequencing results.Conclusion: Maackiain can inhibit the proliferation, cloning, migration and promote the apoptosis of CNE1 cells,and its potential mechanism may be related to the transcriptional changes of P53, P21, Fgf2, Pi3 k and Mapk8 and ot

关 键 词：高丽槐素 鼻咽癌 CNE1细胞 高通量测序 

分 类 号：R739.63[医药卫生—肿瘤]