基于转录组学探讨华蟾酥毒基抑制肝癌细胞增殖的作用机制  

Transcriptomics⁃based exploration on mechanism of action of cinobufagin in inhibiting the proliferation of hepatocellular carcinoma cells

作　　者：孔晨玥 杨文娜 闫秀丽[2] 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)

机构地区：[1]上海中医药大学交叉科学研究院,上海201203 [2]上海中医药大学附属岳阳中西医结合医院消化科,上海200437

出　　处：《上海中医药杂志》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)。

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 rel

关 键 词：华蟾酥毒基 肝癌细胞 转录组学 作用机制 中药研究 

分 类 号：R285[医药卫生—中药学]