八月札乙醇提取物对人肝癌细胞抑制增殖、促进凋亡及转录组学分析  

Transcriptome analysis of ethanol extract of Akebia trifoliate(Thunb.)Koidz on cell proliferation inhibitory functions and apoptosis promotion in human liver cells

作　　者：王春玲[1,2] 文晓东 WANG Chunling;WEN Xiaodong(Key Laboratory of TCM Neuro-Metabolism and Immunopharmacology of Guangxi Education Department,Guangxi University of Chinese Medicine,Nanning 530200,China;College of Pharmacy,Guangxi University ofChinese Medicine,Nanning 530200;Brain Disease Area 1,Ruikang Hospital Affiliated to Guangxi University ofChinese Medicine,Nanning 530011)

机构地区：[1]广西中医药大学广西高校中药神经-代谢及免疫药理重点实验室,南宁530200 [2]广西中医药大学药学院,南宁530200 [3]广西中医药大学附属瑞康医院脑病一区,南宁530011

出　　处：《中国比较医学杂志》2024年第8期60-71,共12页Chinese Journal of Comparative Medicine

基　　金：广西壮族自治区中医药管理局课题(GXZYA20220096);广西中医药大学校级科研项目(2023MS016)。

摘　　要：目的探讨八月札乙醇提取物(ethanol extract of Akebia trifoliate(Thunb.)Koidz,EEATK)对人肝癌细胞增殖、凋亡的影响及可能作用机制。方法体外培养人肝癌细胞(Hep3B和Huh-7),设置空白对照组、索拉菲尼(Sorafenib,5μmol/L)组及EEATK组(0.10、0.15、0.20、0.3 mg/mL),分别给予对应药物干预,采用CCK-8法检测不同分组的药物干预对肝癌细胞活力的影响,筛选最佳作用浓度用于后续实验;采用EdU染色法、克隆形成实验、Annexin V-FITC/PI双染法观察EEATK(0.15 mg/mL)对人肝癌细胞(Hep3B和Huh-7)增殖和凋亡的影响;转录组测序分析EEATK调控Hep3B细胞增殖、凋亡的差异表达基因;使用基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析差异表达基因的功能及富集通路;并联合比较毒物基因组学(Comparative Toxicogenomics Database,CTD)数据库分析与肝癌增殖、凋亡相关基因,应用qRT-PCR验证关键基因表达情况。结果与空白对照组相比,随着EEATK浓度增加,能够显著抑制Hep3B和Huh-7细胞的活力(P<0.01);0.15 mg/mL的EEATK作用肝癌细胞后,EdU阳性细胞率、细胞克隆形成率均显著降低(均P<0.01),同时细胞凋亡率显著增加(P<0.01);Hep3B细胞转录组测序结果分析显示,与空白对照组相比,EEATK引起1577个基因表达发生显著改变(P<0.01),其中表达上调差异基因942个,表达下调差异基因635个。GO功能富集分析发现差异表达基因主要显著富集于胆固醇合成、炎症、细胞外基质中。KEGG通路分析提示EEATK可能主要通过转化生长因子-β(transforming growth factor-β,TGF-β)信号通路及核因子-κB(nuclear factor kappa-B,NF-κB)信号通路发挥抑癌作用。CTD分析及qRT-PCR检测显示EEATK可引起BIRC5、细胞周期蛋白依赖性激酶1(cyclin-dependent kinases 1,CDK1)等细胞凋亡相关基因的表达显著下调(P<0.01),周期蛋白抑制因子1A(cyclin dependent kinase inhibitor 1A,CDKObjective To investigate the effects of ethanol extract of Akebia trifoliate(Thunb.)Koidz(EEATK)on the proliferation and apoptosis of human hepatocellular carcinoma Hep3B and Huh-7 cells and to explore its underlying mechanism.Methods Human Hep3B cells and Huh-7 cells were cultured in vitro and separated into control group,Sorafenib group(5μmol/L),and EEATK groups(0.10 mg/mL,0.15 mg/mL,0.20 mg/mL,0.3 mg/mL)and given the corresponding drug interventions.A CCK-8 assay was used to measure the impact of the different interventions on the proliferation of Hep3B cells and Huh-7 cells to screen the optimal action-inducing concentrations for subsequent experiments.EdU staining assay and colony formation assay were used to explore the effect of EEATK on proliferation,and Annexin V-FITC/PI double-staining assay was applied for apoptotic rate analysis.Transcriptome sequencing(RNA-seq)technology was used to analyze differentially expressed genes(DEGs)related to cell proliferation and apoptosis in the control group and EEATK(0.15 mg/mL)groups of Hep3B cells.DEGs were analyzed for function with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.The Comparative Toxicogenomics Database(CTD)was used to validate the expression of key proteins related to cell proliferation and apoptosis,and the findings were verified by qRT-PCR.Results Compared with the control group,different concentration of EEATK significantly inhibited the activity of Hep3B and Huh-7 cells(P<0.01).Hep3B cells were treated with 0.15 mg/mL EEATK,the EdU-positive cell rate and clone formation rate significantly decreased(all P<0.01).At the same time,the apoptotic rate of the EEATK group significantly increased(P<0.01).Transcriptome sequencing of Hep3B cells showed that EEATK induced significant changes in the expression of 1577 genes(P<0.01),of which 942 were up-regulated and 635 were down-regulated compared with the control group.GO functional enrichment analysis revealed that the DEGs were mainly enriched for cholesterol synthe

关 键 词：八月札 肝癌 增殖 凋亡 转录组学 

分 类 号：R-33[医药卫生]