目的 探讨白杨素在体内外对人结肠癌HCT-116细胞侵袭与转移作用及相关机制。方法 本实验应用CCK-8法检测白杨素对HCT-116细胞增殖能力的抑制作用。利用划痕和Transwell法检测白杨素干预HCT-116细胞后对细胞迁移和侵袭能力的作用。通过Western blot检测白杨素干预后对HCT-116细胞中上皮间质转化(EMT)相关蛋白E-cadherin、N-cadherin、Vimentin和Snail表达水平的影响,并检测白杨素对HCT-116细胞内Yes相关蛋白(YAP)蛋白表达的作用。利用siRNA干扰实验考察YAP低表达对白杨素在HCT-116细胞侵袭作用和EMT进程中的影响。构建体内实验性肺转移模型考察白杨素对HCT-116细胞体内转移的作用。结果 白杨素从8 μmol·L-1开始能够显著抑制HCT-116细胞的增殖,8 μmol·L-1以下对HCT-116细胞增殖没有显著抑制作用。划痕和Transwell实验显示,白杨素(1、2和4 μmol·L-1)能够剂量依赖性的抑制HCT-116细胞的迁移与侵袭。Western blot结果显示,白杨素能够上调E-cadherin的蛋白表达,下调N-cadherin,Vimentin及转录因子Snail的蛋白表达。同时还发现白杨素能够下调YAP蛋白表达水平,而利用siRNA下调YAP后能显著逆转白杨素对HCT-116细胞迁移与侵袭的抑制作用,并能逆转白杨素对HCT-116细胞EMT过程的促进作用。体内肺转移实验显示,白杨素显著抑制了HCT-116在肺上转移结节。结论 本实验表明,白杨素能够在体内外抑制人结肠癌HCT-116细胞的侵袭与转移能力,其作用机制与抑制YAP的蛋白表达,下调EMT过程相关。
Abstract
OBJECTIVE To investigate the effect and related mechanism of chrysin on the invasion and metastasis of human colon cancer HCT-116 cells in vitro and in vivo. METHODS CCK-8 was used to detect the inhibitory effect of chrysin on the proliferation of HCT-116 cells. The effects of chrysin on cell migration and invasion were detected by scratch and Transwell. Western blot was used to detect the effects of chrysin on the expression levels of EMT-related proteins E-cadherin, N-cadherin, Vimentin, and Snail in HCT-116 cells and the effect of chrysin on the expression of YAP protein in HCT-116 cells. SiRNA interference assay was used to investigate the effect of YAP low expression on chrysin in HCT-116 cell invasion and EMT process. Experimental lung metastasis model was constructed to investigate the effect of chrysin on the metastasis of HCT-116 cells in vivo. RESULTS Chrysin could significantly inhibit the proliferation of HCT-116 cells at concentration higher than 8 μmol·L-1, and had no significant cytotoxicity on HCT-116 cells when the concentration was below 8 μmol·L-1. Chrysin of 1, 2 and 4 μmol·L-1 inhibited the migration and invasion of HCT-116 cells in a dosage-dependent manner. Western blot results showed that chrysin could up-regulate the protein expression of E-cadherin and down-regulate the protein expression of N-cadherin, Vimentin, and the transcription factor Snail. It was also found that chrysin could down-regulate the protein level of YAP, while down-regulation of YAP could significantly reverse the inhibition of chrysin on migration and invasion of HCT-116 cells and the promotion effect of chrysin on the EMT process of HCT-116 cells by siRNA. The lung metastasis experiments showed that chrysin significantly inhibited the pulmonary metastasis nodules of HCT-116 cells. CONCLUSION This study shows that chrysin can inhibit the invasion and metastasis of HCT-116 cells in vitro and in vivo, and the mechanism is related to the inhibition of YAP and down-regulation of EMT.
关键词
白杨素 /
结肠癌 /
上皮间质转化 /
细胞侵袭 /
肿瘤转移 /
Yes相关蛋白
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Key words
chrysin /
colon cancer /
epithelial-mesenchymal transition /
tumor invasion /
tumor metastasis /
Yes-associated protein
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中图分类号：
R965
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参考文献
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脚注
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基金
南京医科大学科技发展基金项目资助(NMUB2018315);南京市医学科技发展项目资助(YKK15090)
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