基于转录组学分析肿节风总黄酮抑制白血病K562细胞生长的作用机制  

Transcriptomic Analysis of Inhibitory Mechanism of Total Flavonoids From Sarcandrae Herba on the Growth of Leukemia K562 Cells

作　　者：尚广彬[1,2] 柳歌 孙慧娟 陈中 卢晓南[3] 严小军 伍庆华 SHANG Guangbin;LIU Ge;SUN Huijuan;CHEN Zhong;LU Xiaonan;YAN Xiaojun;WU Qinghua(Research Center for Differentiation and Development of Basic Theory of TCM,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Jiangxi Province Key Laboratory of TCM Etiopathogenisis,Nanchang 330004 Jiangxi,China;School of Traditional Chinese Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China)

机构地区：[1]江西中医药大学中医基础理论分化发展中心,江西南昌330004 [2]江西省中医病因生物学重点实验室,江西南昌330004 [3]江西中医药大学中医学院,江西南昌330004

出　　处：《中药新药与临床药理》2022年第11期1445-1452,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：江西省教育厅科学技术研究项目(GJJ190676);国家自然科学基金项目(81960743)。

摘　　要：目的 利用转录组学技术结合体外实验揭示肿节风总黄酮抑制白血病K562细胞生长的作用机制。方法 将K562细胞分为空白对照组、二甲基亚砜(DMSO)对照组及肿节风总黄酮低、中、高剂量组(25、50、100μg·mL^(-1)),干预48 h后采用化学发光法检测K562细胞活力。运用转录组测序(RNA-seq)技术对DMSO对照组和肿节风总黄酮高剂量组(100μg·mL^(-1))K562细胞进行差异表达基因(DEGs)分析;并对差异表达基因进行基因本体论(GO)功能及京都基因与基因组百科全书(KEGG)通路富集分析。采用Western Blot法和流式细胞术验证筛选出的MAPK通路中关键蛋白的表达情况,以及细胞周期变化。结果 与DMSO对照组比较,肿节风总黄酮低、中、高剂量能够显著抑制K562细胞活力(P<0.01)。转录组分析共筛选出989个差异表达基因,其中654个上调、335个下调。KEGG通路富集分析显示,肿节风总黄酮对K562细胞的作用与MAPK信号通路、神经活性配体-受体相互作用、补体和凝血级联途径高度相关。与DMSO对照组比较,高剂量肿节风总黄酮能够显著降低K562细胞的p-JNK、CDC20蛋白表达水平(P<0.01),升高P53、CDKN1A蛋白表达水平(P<0.01),明显阻滞K562细胞在G0/G1期(P<0.01)。结论 肿节风总黄酮可能通过降低MAPK信号通路中JNK蛋白磷酸化水平,上调P53、CDKN1A蛋白表达,下调CDC20蛋白表达,阻滞细胞周期,进而抑制白血病K562细胞的生长活性。Objective To utilize transcriptomic techniques combined with in vitro experiments to reveal the mechanism of total flavonoids from Sarcandrae Herba in the inhibition of leukemia K562 cells growth.Methods K562 cells were divided into the blank control group,the dimethyl sulfoxide(DMSO)control group and the low-,medium-and high-dose groups(25,50 and 100μg·mL^(-1))of total flavonoids from Sarcandrae Herba,and the viability of K562 cells was detected by chemiluminescence after 48 hours of intervention.Differential expressed genes(DEGs) analysis was performed on K562 cells in the DMSO control group and total flavonoids from Sarcandrae Herba high-dose group(100μg·mL^(-1))using transcriptome sequencing(RNA-seq)technology;and differential genes were analyzed for Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.Western Blot and flow cytometry were used to validate the expression of key proteins in the screened MAPK pathway,as well as cell cycle changes.Results Compared with DMSO control group,low-,medium-and high-dose groups of total flavonoids from Sarcandrae Herba could significantly inhibit the activity of K562 cells(P<0.01).Transcriptomic analysis identified 989 DEGs,of which 654 were up-regulated and 335 were downregulated.KEGG pathway enrichment analysis showed that the effects of total flavonoids from Sarcandrae Herba on K562 cells were highly correlated with MAPK signaling pathway,neuroactive ligand-receptor interaction,complement and coagulation cascade pathways.Compared with the DMSO control group,high-dose of total flavonoids from Sarcandrae Herba significantly reduced protein expression levels of p-JNK and CDC20 in K562 cells(P<0.01),increased protein expression levels of P53 and CDKN1A(P<0.01),and significantly blocked K562cells in the G0/G1 phase(P<0.01).Conclusion The total flavonoids from Sarcandrae Herba may inhibit the growth activity of leukemia cells by reducing the phosphorylation level of JNK protein in the MAPK signaling pathway,upregulating protein e

关 键 词：肿节风总黄酮 白血病K562细胞 转录组学 MAPK信号通路 细胞周期 

分 类 号：R285.5[医药卫生—中药学] R857.3[医药卫生—中医学]