[Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway].
[Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway].
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DOI：
10.19540/j.cnki.cjcmm.20200108.401
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发表时间：
2020-04-01
期刊：
影响因子：
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通讯作者：
Xu, Hai-Bo
中科院分区：
文献类型：
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作者：
Lei, Nan;Xiong, Si-Hui;Xu, Hai-Bo
本研究旨在观察灯盏乙素对结肠癌干细胞分化的抑制作用，探讨灯盏乙素抑制结肠癌干细胞分化的hedgehog信号机制。采用三维细胞培养法观察灯盏乙素对HT-29细胞来源的癌干细胞样细胞（HT-29 CSC）体外生长的影响。软琼脂集落形成实验检测灯盏乙素对HT-29 CSC细胞转化的影响。采用胎牛血清体外诱导干细胞分化，观察灯盏乙素对HT-29 CSC细胞分化的影响。采用实时荧光定量聚合酶链反应（qRT-PCR）检测灯盏乙素对HT-29 CSC细胞Lgr 5、c-Myc、CK 20和Nanog mRNA表达的影响。Western blot检测灯盏乙素对HT-29 CSC细胞c-Myc、Gli 1和Lgr 5蛋白表达的影响。将HT-29 CSC细胞接种于裸鼠皮下，观察灯盏乙素对裸鼠体重及HT-29 CSC肿瘤生长的影响。qRT-PCR检测灯盏乙素对肿瘤组织中CD 133、Lgr 5、Gli 1、Ptch 1、c-Myc、Ki-67、CK 20和Nanog mRNA水平的影响。Western blot和免疫组化检测灯盏乙素对肿瘤组织中c-Myc、Gli 1、Lgr 5、CD 133和Ki-67蛋白表达的影响。体外实验表明，灯盏乙素可抑制HT-29 CSC细胞的生长、转化和分化，显著下调HT-29 CSC细胞中Lgr 5、c-Myc、CK 20和Nanog的mRNA水平，以及HT-29 CSC细胞中c-Myc、Gli 1和Lgr 5的蛋白表达水平。此外，动物实验表明灯盏乙素显著抑制裸鼠皮下移植瘤的生长，下调裸鼠皮下移植瘤CD 133、Lgr 5、Gli 1、Ptch 1、c-Myc、Ki-67、CK 20和Nanog的mRNA表达以及c-Myc、Gli 1、Lgr 5、CD 133和Ki-67的蛋白水平。灯盏乙素在体内外均能抑制结肠癌干细胞的分化，其机制可能与下调hedgehog信号通路的活性有关。
The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.