摘要
目的观察华蟾酥毒基(CBF)对肝癌细胞增殖的抑制作用,采用转录组学测序技术揭示相关基因及信号通路,探讨CBF抗肝细胞癌(HCC)的可能作用机制。方法用不同浓度的CBF(0、2、4、8、16和32μmol·L^(-1))处理人HCC细胞SMMC-7721和JHH7,时间梯度为24、48和72 h,WST-1试剂盒检测细胞增殖情况。根据WST-1试剂盒检测结果,用CBF 2μmol·L^(-1)干预SMMC-7721和JHH7细胞24 h,然后进行转录组学基因表达测序分析差异表达基因,并行生物信息学分析。基于文献报道及TCGA数据库观察部分差异表达基因在HCC组织中的表达情况,采用实时荧光定量逆转录聚合酶链式反应(RT-qPCR)技术检测并验证CBF靶基因的表达情况。结果①CBF对SMMC-7721和JHH7细胞具有抑制增殖作用,且呈浓度-时间依赖性。②转录组学测序结果显示,经CBF干预后,SMMC-7721细胞获得差异表达基因共计578个(其中上调基因309个、下调基因269个),JHH7细胞获得差异表达基因共计611个(其中上调基因349个、下调基因262个),两组细胞获得的交集基因共有106个(差异倍数>2,P<0.05)。③生物信息学分析显示,激活转录因子3(ATF3)、羰基还原酶1(CBR1)、细胞骨架相关蛋白4(CKAP4)、生长停滞和DNA损伤可诱导蛋白β(GADD45B)、钾二孔域通道亚科K成员5(KCNK5)、起源识别复合亚基5(ORC5)基因的转录水平在HCC组织中呈异常表达并与患者预后相关。④RT-qPCR结果显示,CBF能促进ATF3、KCNK5和GADD45B基因的表达,抑制CBR1、CKAP4和ORC5基因的表达,与测序结果一致。结论CBF具有抑制HCC细胞增殖的作用,其机制可能与调控ATF3、KCNK5、GADD45B、CBR1、CKAP4和ORC5基因表达有关。
Objective To investigate the inhibitory effect of cinobufagin(CBF)on the proliferation of hepatocellular carcinoma(HCC)cells,reveal the related genes and signaling pathways by transcriptomic sequencing,and explore the possible mechanism of action of anti-HCC effect of CBF.Methods SMMC-7721 and JHH7 cells were treated with CBF at different concentrations(0,2,4,8,16 and 32μmol·L^(-1))at 24,48 and 72 h.WST-1 assay was used to assess the proliferation rate of HCC cells.Based on the results of WST-1 assay,SMMC-7721 and JHH7 cells were intervened with CBF 2μmol·L^(-1) for 24 h,differentially expressed genes were analyzed with transcriptomic gene expression sequencing,and bioinformatics analysis was performed.Based on literature reports and the TCGA database,the expression of a few differently expressed mRNAs in HCC tissues was discovered.The reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect and validate the expression of CBF target genes.Results①CBF had a proliferation-inhibitory effect on SMMC-7721 and JHH7 cells in a concentration-time dependent manner.②Transcriptomic sequencing results showed that in CBF-treated SMMC-7721 cells,a total of 578 differentially expressed genes(309 up-regulated genes and 269 down-regulated genes)were obtained;in CBF-treated JHH7 cells,a total of 611 differentially expressed genes(349 up-regulated genes and 262 down-regulated genes)were obtained;and a total of 106 intersecting genes were obtained in the two groups of cells(fold change>2,P<0.05).③Bioinformatics analysis showed that the transcript levels of ATF3,CBR1,CKAP4,GADD45B,KCNK5 and ORC5 were aberrantly expressed in HCC tissues and correlated with patient prognosis.④The RT-qPCR results showed that CBF promoted the expression of ATF3,KCNK5 and GADD45B genes and inhibited the expression of CBR1,CKAP4 and ORC5 genes in SMMC-7721 and JHH7 cells,which was consistent with the sequencing results.Conclusion CBF can inhibit the proliferation of HCC cells,and the mechanism may be related to the regulation of ATF3,KCNK5,GADD45B,CBR1,CKAP4 and ORC5 expression.
作者
孔晨玥
杨文娜
闫秀丽
张辉
KONG Chenyue;YANG Wenna;YAN Xiuli;ZHANG Hui(Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,ShanghaiUniversity of Traditional Chinese Medicine,Shanghai 200437,China)
出处
《上海中医药杂志》
CSCD
2023年第8期49-56,共8页
Shanghai Journal of Traditional Chinese Medicine
基金
国家自然科学基金项目(82074101)
上海市卫健委临床研究专项(202040486)。
关键词
华蟾酥毒基
肝癌细胞
转录组学
作用机制
中药研究
cinobufagin
hepatocellular carcinoma cells
transcriptomics
mechanism of action
research of traditional Chinese herbal medicine
