基于转录组与体外实验揭示泽泻醇B抑制神经胶质瘤细胞增殖作用的机制  

Study on the Mechanism of Inhibitory Effect of Alismol B on Glioma Cell Proliferation Based on Transcriptome and In Vitro Experiments

作　　者：文香月 徐小妹 张亚敏 卢雪花 李丽莎 江若红 林文津 WEN Xiang-yue;XU Xiao-mei;ZHANG Ya-min;LU Xue-hua;LI Li-sha;JIANG Ruo-hong;LIN Wen-jin(Fujian Academy of Medical Sciences/Fujian Key Laboratory of Medical Testing,Fuzhou 350001,China;College of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou 350001,China)

机构地区：[1]福建省医学科学研究院/福建省医学测试重点实验室,福建福州350001 [2]福建中医药大学药学院,福建福州350001

出　　处：《中药材》2023年第8期2030-2038,共9页Journal of Chinese Medicinal Materials

基　　金：福建省公益科研院所基本专项(2021R1021001);福建省医学创新课题(2021CXA038);福建省中医药科研项目(2021zyyj71)。

摘　　要：目的:分析人脑胶质瘤细胞系T98G对泽泻醇B的反应及其作用机制。方法:泽泻醇B处理人脑胶质瘤细胞T98G后,通过细胞计数试剂盒(CCK-8)分析细胞活力,划痕、侵袭和迁移实验评估细胞运动能力。用10、20μmol/L泽泻醇B处理T98G细胞,利用高通量测序技术获得基因表达谱,并对差异表达基因(DEGs)进行生物信息学分析。结合转录组测序分析结果和细胞表型,筛选出肿瘤相关靶点进行qPCR和Western Blot验证。结果:体外研究显示与空白对照组比较,泽泻醇B能有效抑制人脑胶质瘤细胞T98G增殖、侵袭和迁移。转录组测序结果显示泽泻醇B 10μmol/L组中,有差异表达基因3 235个,其中上调基因1 502个,下调基因1 733个;泽泻醇B 20μmol/L组中,有差异基因4 957个,其中上调基因2 362个,下调基因2 595个。KEGG通路结果显示,泽泻醇B 10、20μmol/L组差异表达基因共富集通路包括MAPK信号通路、细胞周期、肿瘤microRNAs、P53信号通路、DNA复制,其中MAPK信号通路差异基因富集数量最多。qPCR显示DEGs mRNA表达量变化与转录组测序基本一致。结论:泽泻醇B能有效抑制神经胶质瘤细胞增殖、侵袭及迁移,其发挥抗细胞增殖作用机制可能与抑制MAPK信号通路上GRB2、AKT1、ERK1/2等关键靶点表达和磷酸化有关;其抑制T98G细胞侵袭和迁移的能力可能与下调MMP1和MMP3蛋白表达,升高TIMP3蛋白表达有关。Objective:To study the reaction of human glioma cell line T98G to alismol B and its mechanism.Methods:After treating human glioma cells T98G with alismol B,cell activity was analyzed by cell counting kit(CCK-8),and cell motility was evaluated by scratch,invasion and migration assay.T98G cells were treated with 10 and 20μmol/L alismol B,and the gene expression profiles were obtained by high-throughput sequencing technology,and bioinformatics analysis of differentially expressed genes(DEGs)was performed.Combined with transcriptome sequencing analysis results and cell phenotypes,tumor-related targets were screened for qPCR and Western Blot verification.Results:In vitro studies showed that alismol B could effectively inhibit the proliferation,invasion and migration of human glioma cells T98G compared with the normal control group.Transcriptome sequencing showed that there were 3235 differentially expressed genes in the 10μmol/L alismol B group,including 1502 up-regulated genes and 1733 down-regulated genes.There were 4957 differential genes in the 20μmol/L alismol B group,including 2362 up-regulated genes and 2595 down-regulated genes.The results of KEGG pathway showed that the co-enrichment pathways of differentially expressed genes in 10 and 20μmol/L alismol B groups included MAPK signaling pathway,cell cycle,tumor microRNAs,P53 signaling pathway,DNA replication,among which MAPK signaling pathway had the highest concentration of differentially expressed genes.qPCR showed that the expression of DEGs mRNA was basically consistent with transcriptome sequencing.Conclusion:Alismol B can effectively inhibit the proliferation,invasion and migration of glioma cells.The mechanism of its anti-proliferation effect may be related to the inhibition of the expression and phosphorylation of GRB2,AKT1,ERK1/2 and other key targets in MAPK signaling pathway.The inhibition on invasion and migration of T98G cells may be related to the down-regulation of MMP1 and MMP3 protein expressions and the increase of TIMP3 protein expression

关 键 词：人脑胶质瘤细胞T98G MAPK信号通路 转录组测序 泽泻醇B 

分 类 号：R285.5[医药卫生—中药学]