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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
DOI: 10.12449/JCH240919
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。
Mechanism of action of cinobufotalin in inhibiting lung metastasis of hepatocellular carcinoma by regulating AKT-mediated epithelial-mesenchymal transition in a nude mouse model
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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。 Abstract:Objective To investigate the effect and mechanism of cinobufotalin in inhibiting hepatocellular carcinoma （HCC） metastasis by regulating epithelial-mesenchymal transition （EMT）. Methods A total of 36 male BALB/c nude mice， aged 6 weeks， were given injection of MHCC97H cells via the caudal vein to establish a model of HCC lung metastasis， and then the mice were randomly divided into high-and low-dose cinobufotalin groups and control group. Since the day of modeling， the mice in the high-and low-dose cinobufotalin groups were given intraperitoneal injection of cinobufotalin at a dose of 120 μL/kg and 60 μL/kg， respectively， and those in the control group were given intraperitoneal injection of normal saline， twice a week. After 8 weeks， HE staining was performed for lung tissue to measure the lung metastasis rate of HCC. MHCC97H cells were treated with high-dose （2.5 μL/mL） or low-dose （5 μL/mL） cinobufotalin for 24 hours， and wound healing assay， RT-PCR， and Western blot were used to measure cell migration ability and the expression of EMT-related molecules. MHCC97H cells were induced in a simulated hypoxic environment with CoCl2 incubation， with high- and low-dose cinobufotalin added for intervention， and wound healing assay and Western blot were used to investigate the effect of cinobufotalin on cell migration ability and EMT induced by hypoxia. Transcriptome analysis was used to investigate the effect mechanism of cinobufotalin on MHCC97H cells， and Western blot was used to observe the effect of cinobufotalin on the expression levels of protein kinase B （AKT） and phosphorylated AKT （P-AKT） in MHCC97H cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups； the independent-samples t test was used for comparison of categorical data between two groups. Results Compared with the control group， the cinobufotalin group had a significant reduction in the lung metastasis rate of HCC. Compared with the control group， cinobufotalin intervention reduced the wound healing rate of MHCC97H cells， upregulated the expression of epithelial-type molecules （t=2.860， P<0.05）， and downregulated the expression of EMT transcription factors （EMT-TFs） and mesenchymal molecules （t=3.545， 2.022， 2.852， and 2.341， all P<0.05）. Hypoxia induction upregulated the wound healing rate of MHCC97H cells and the expression levels of mesenchymal molecules and EMT-TFs （P<0.05）， and cinobufotalin intervention reversed EMT change and inhibited wound healing （P<0.05）. The transcriptome analysis of MHCC97H cells showed significant gene differences between the cinobufotalin group and the control group， and cinobufotalin mainly affected the expression of genes associated with tumor， metabolism， immunity， and signal transduction， with the largest number of differentially expressed genes in the AKT signal transduction pathway. Further measurement showed that cinobufotalin intervention downregulated the expression levels of AKT， P-AKT， and P-AKT/AKT in MHCC97H cells （t=2.434， 3.401， and 2.258， all P<0.05）. Conclusion Cinobufotalin can inhibit the metastasis of HCC， especially hypoxia-induced HCC metastasis， and regulation of EMT mediated by the AKT signal transduction pathway in HCC cells might be one of its mechanisms of action. -
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。
图 1 华蟾素对裸鼠肝癌肺转移的影响
Figure 1. The effect of Cinobufotalin on lung metastasis of HCC in nude mice
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。
图 2 华蟾素对肝癌细胞迁移能力的影响
Figure 2. The effect of Cinobufotalin on the migration ability of HCC cells
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。
图 3 华蟾素对肝癌细胞EMT相关分子表达水平的影响
Figure 3. The effect of Cinobufotalin on the expression of EMT-related molecules in HCC cells
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。
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
Figure 4. The effect of Cinobufotalin on hypoxia-induced cell migration ability and EMT of HCC cells
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。
图 5 华蟾素干预的HCC细胞转录组学分析
Figure 5. Seq-RNA analysis of Cinobufotalin -treated HCC cells
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