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华蟾素调控AKT介导的上皮间质转化抑制肝细胞癌肺转移裸鼠模型的作用机制

杨悦 续嗣钰 王珏 杜施霖 张春蕾 宋海燕

引用本文:
Citation:

华蟾素调控AKT介导的上皮间质转化抑制肝细胞癌肺转移裸鼠模型的作用机制

DOI: 10.12449/JCH240919
基金项目: 

上海市青年科技英才扬帆计划项目 (20YF1450000);

龙华医院科技创新项目 (CX202205);

上海市卫生健康委卫生行业临床研究专项 (202040223)

伦理学声明:本研究方案于2019年4月18日经由上海中医药大学附属龙华医院实验动物伦理委员会审批,批号:LHERAW-19017,符合实验室动物管理与使用准则。
利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:杨悦、续嗣钰、王珏、杜施霖参与收集数据,资料分析;杨悦负责撰写论文;张春蕾、宋海燕负责课题设计,指导撰写文章并最后定稿。
详细信息
    通信作者:

    张春蕾, clzhang213@163.com (ORCID: 0000-0002-3934-7816)

    宋海燕, songhy@126.com (ORCID: 0000-0003-2155-8110)

Mechanism of action of cinobufotalin in inhibiting lung metastasis of hepatocellular carcinoma by regulating AKT-mediated epithelial-mesenchymal transition in a nude mouse model

Research funding: 

Shanghai Youth Technology Talents Sailing Program (20YF1450000);

Longhua Hospital Technology Innovation Project (CX202205);

Clinical Research Special Project of Shanghai Municipal Health Commission (202040223)

More Information
    Corresponding author: ZHANG Chunlei, clzhang213@163.com (ORCID: 0000-0002-3934-7816); SONG Haiyan, songhy@126.com (ORCID: 0000-0003-2155-8110)
  • 摘要:   目的  研究华蟾素通过调控肝细胞癌(HCC)上皮间质转化(EMT)抑制HCC转移的作用和机制。  方法  将36只6周龄雄性BALB/c裸鼠尾静脉注射MHCC97H细胞建立肝癌肺转移瘤模型,随机分为华蟾素高、低剂量组和对照组。建模当日起分别腹腔注射华蟾素120 μL/kg、60 μL/kg或生理盐水,每周2次。8周后取肺组织行HE染色检测肝癌肺转移率。MHCC97H细胞用华蟾素高、低剂量(2.5 μL/mL、5 μL/mL)干预,通过划痕实验、RT-PCR以及Western Blot检测细胞迁移能力和EMT相关分子的表达。用CoCl2孵育模拟低氧环境诱导MHCC97H细胞,同时加入高、低剂量华蟾素干预,通过划痕实验和Western Blot检测华蟾素对低氧诱导的细胞迁移能力和EMT的影响。使用转录组学分析华蟾素对MHCC97H细胞的效应机制。用Western Blot检验华蟾素干预对MHCC97H细胞的蛋白激酶B(AKT)、磷酸化AKT(P-AKT)表达水平的影响。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验,两组间比较采用成组t检验。  结果  华蟾素干预组裸鼠较对照组肝癌肺转移率下降。与对照组相比,华蟾素干预使MHCC97H细胞划痕愈合率减小、上皮型分子表达上调(t=2.860,P<0.05),并使EMT转录因子和基质型分子下调(t值分别为3.545、2.022、2.852、2.341,P值均<0.05)。低氧诱导上调MHCC97H细胞划痕愈合率和基质型分子、EMT转录因子表达水平(P值均<0.05),华蟾素干预逆转EMT变化并抑制划痕愈合(P值均<0.05)。肝癌细胞转录组学分析显示,华蟾素组与对照组存在显著的基因差异,华蟾素主要影响了肿瘤、代谢、免疫和信号传导相关基因表达,其中AKT信号转导通路中的差异基因数量最多。进一步检测发现华蟾素干预可下调HCC 细胞AKT、P-AKT和P-AKT/AKT的水平(t值分别为2.434、3.401、2.258,P值均<0.05)。  结论  华蟾素可抑制肝癌转移,尤其对于低氧环境诱导的肝癌转移具有显著抑制作用,调控AKT信号转导通路介导的HCC细胞EMT可能是其部分作用机制。

     

  • 图  1  华蟾素对裸鼠肝癌肺转移的影响

    注: a,肺组织HE染色(×4);b:肺转移率;c:血清ALT活力(U/L);d:血清肌酐含量(μmol/L)。Control:对照组;HCS-L:低剂量华蟾素组;HCS-H:高剂量华蟾素组。箭头标示HCC转移灶。

    Figure  1.  The effect of Cinobufotalin on lung metastasis of HCC in nude mice

    图  2  华蟾素对肝癌细胞迁移能力的影响

    注: a: 划痕愈合图(×4);b: 3组划痕愈合率比较。Control:对照组;HCS-L:低剂量华蟾素组;HCS-H:高剂量华蟾素组。

    Figure  2.  The effect of Cinobufotalin on the migration ability of HCC cells

    图  3  华蟾素对肝癌细胞EMT相关分子表达水平的影响

    注: a,EMT相关分子mRNA表达水平;b,EMT相关分子蛋白表达水平;c,蛋白表达分析。Control:对照组;HCS-L:低剂量华蟾素组;HCS-H:高剂量华蟾素组; HCS:华蟾素组。

    Figure  3.  The effect of Cinobufotalin on the expression of EMT-related molecules in HCC cells

    图  4  华蟾素对低氧环境诱导的肝癌细胞迁移能力和EMT的影响

    注: a,划痕愈合实验图;b,划痕愈合率;c,EMT相关分子蛋白条带;d,EMT相关分子蛋白分析。Control:对照组;CoCl2:模型组;HCS-L:低剂量华蟾素组;HCS-H:高剂量华蟾素组。

    Figure  4.  The effect of Cinobufotalin on hypoxia-induced cell migration ability and EMT of HCC cells

    图  5  华蟾素干预的HCC细胞转录组学分析

    注: a,对照组和华蟾素组之间的差异基因聚类热图;b,表达量差异火山图;c,核心靶点的 KEGG 富集分析图。

    Figure  5.  Seq-RNA analysis of Cinobufotalin -treated HCC cells

    图  6  华蟾素对肝癌细胞AKT信号通路的调控作用

    注: a. 蛋白条带图;b:蛋白表达分析。Control:对照组;HCS:华蟾素组。

    Figure  6.  The regulatory effect of cinobufotalin on AKT signaling pathway in HCC cells

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