猫须草总黄酮对人肾透明细胞癌786-O细胞凋亡的影响及转录组学分析  

Effect of total flavonoids from Clerodendranthus Spicatus on apoptosis of human renal clear cell carcinoma 786⁃O cells and transcriptomic analysis

作　　者：廖宏亮 郭云涛 唐帅男 陈双剑 朱云 赵德志 姜昕 马端 LIAO Hongliang;GUO Yuntao;TANG Shuainan;CHEN Shuangjian;ZHU Yun;ZHAO Dezhi;JIANG Xin;MA Duan(Jiangxi University of Chinese Medicine,Nanchang 330103,Jiangxi,China;Zhongke Gene Biotechnology(Jiangsu)Co,Ltd.,Nantong 226133,Jiangsu,China;Yangtze Delta Drug Advanced Research Institute,Nantong 226133,Jiangsu,China)

机构地区：[1]江西中医药大学,江西南昌330103 [2]中科基因生物科技(江苏)有限公司,江苏南通226133 [3]长三角药物高等研究院,江苏南通226133

出　　处：《上海中医药大学学报》2023年第3期48-56,共9页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：国家重点研发计划资助项目(2021YFC270101)。

摘　　要：目的:探讨猫须草总黄酮对人肾透明细胞癌786-O细胞凋亡的影响及其机制。方法:用不同浓度(0、25、50、100、200、400µg/mL)的猫须草总黄酮干预人肾透明细胞癌786-O细胞,采用CCK-8法检测细胞活力,采用膜联蛋白V/碘化丙啶(Annexin V/PI)染色检测细胞凋亡。786-O细胞分别用0、139.9µg/mL猫须草总黄酮干预后,采用二代高通量测序平台(Illumina Novaseq 6000)对细胞进行RNA-seq建库测序分析,获得通路相关基因,并采用RT-qPCR和Western blot进行验证。结果:①CCK-8检测结果表明,猫须草总黄酮处理人肾透明细胞癌786-O细胞后,细胞活力呈时间及浓度依赖性降低;猫须草总黄酮呈浓度依赖性地促进786-O细胞凋亡。②通过转录组测序共鉴定出2047个差异基因,其中有773个上调,1274个下调。基因本体论(GO)富集分析显示,差异基因主要涉及细胞应激反应、凋亡信号通路调控、外源性凋亡信号通路等过程;京都基因和基因组百科全书(KEGG)富集分析显示,差异基因主要涉及细胞凋亡、代谢等。③通过RT-qPCR和Western blot验证几个相关基因的mRNA和蛋白表达水平,与转录组测序的趋势一致(P<0.01)。结论:通过RNA-seq测序数据分析,发现猫须草总黄酮可能通过肿瘤抑制蛋白p53(p53)、丝裂原活化蛋白激酶(MAPK)、核因子-κB(NF-κB)等信号通路诱导人肾透明细胞癌786-O细胞凋亡。Objective:To explore the mechanism of total flavonoids from Clerodendranthus Spicatus on the apoptosis of human renal clear cell carcinoma 786-O cells.Methods:Human renal clear cell carcinoma 786-O cells were treated with different concentrations(0,25,50,100,200,400µg/mL)of total flavonoids from Clerodendranthus Spicatus.Cell viability was detected by CCK-8 assay,and cell apoptosis was measured by Annexin V/propidium iodide(PI)staining.RNA-seq library sequencing analysis was carried out through Illumina Novaseq 6000 platform on cells treated with 0 and 139.9µg/mL of total flavonoids from Clerodendranthus Spicatu.Pathway-related genes were obtained and verified by RT-qPCR and Western blot.Results:①CCK-8 results showed that the viability of human renal clear cell carcinoma 786-O cells decreased in a time and concentration-dependent manner after treated with total flavonoids from Clerodendranthus Spicatu.And total flavonoids from Clerodendranthus Spicatu induced apoptosis of 786-O cells in a concentrationdependent manner.②A total of 2047 differential genes were identified by transcriptome sequencing,among which 773 were up-regulated and 1274 were down-regulated.Gene Ontology(GO)enrichment analysis showed that the differential genes were mainly involved in processes such as cellular stress response,apoptotic signaling pathway regulation,and exogenous apoptotic signaling pathway.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the differential genes were mainly involved in cell apoptosis and metabolism.③The mRNA and protein expression levels of several related genes were verified by RT-qPCR and Western blot,which was consistent with the trend of transcriptome sequencing(P<0.01).Conclusion:RNA-seq sequencing data analysis showed that total flavonoids from Clerodendranthus Spicatu may induce apoptosis of human renal clear cell carcinoma 786-O cells through signaling pathways such as tumor suppressor protein p53(p53),mitogen-activated protein kinase(MAPK),and nuclear factor-κB(NF-κB)

关 键 词：肾茶 猫须草总黄酮 肾透明细胞癌 细胞凋亡 转录组测序 信号通路 

分 类 号：R285[医药卫生—中药学]