天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (5): 535-539.doi: 10.11958/20150385

• 专题研究-消化疾病 • 上一篇    下一篇

缺氧诱导因子-1α对结直肠癌细胞上皮间质转化及 侵袭迁移的影响

吴丽丽 1， 孙慧誌 1， 孙冉 2， 赵楠 1,3， 王勇 1， 古强 1,3， 董学易 1,3， 刘芳 1,3， 孙保存 1,3,4△   

  1. 1天津医科大学 （邮编 300070）； 2天津市南开医院； 3天津医科大学总医院； 4天津医科大学肿瘤医院
  • 收稿日期:2015-12-09 修回日期:2016-02-01 出版日期:2016-05-15 发布日期:2016-05-18
  • 通讯作者: △通讯作者 E-mail:sunbaocun@aliyun.com E-mail:wulili0106@163.com
  • 作者简介:吴丽丽 （1989）， 女， 硕士在读， 主要从事肿瘤血管生成拟态的分子调控机制研究
  • 基金资助:
    国家自然科学基金重点项目 （81230050）； 国家自然科学基金面上项目 （81572872）

Effects of HIF-1α on epithelial-mesenchymal transition, invasion and migration in colorectal cancer cells

WU Lili1, SUN Huizhi1, SUN Ran2, ZHAO Nan1,3, WANG Yong1, GU Qiang1,3, DONG Xueyi1,3, LIU Fang1,3, SUN Baocun1,3,4△   

  1. 1 Tianjin Medical University, Tianjin 300070， China； 2 Tianjin Nan Kai Hospital； 3 Tianjin Medical University General Hospital； 4 Tianjin Medical University Cancer Institute and Hospital
  • Received:2015-12-09 Revised:2016-02-01 Published:2016-05-15 Online:2016-05-18
  • Contact: △Corresponding Author E-mail:sunbaocun@aliyun.com E-mail:wulili0106@163.com

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摘要： 摘要： 目的 探讨缺氧是否能促进不同分化程度结直肠癌细胞上皮间质转化 （EMT）， 并分析缺氧对结直肠癌细 胞侵袭、 迁移的影响。方法 分别选取 HCT116（低度分化）和 HT-29（高度分化）结直肠腺癌细胞。观察 0、 10、 25、 50、 100 及 150 mg/L 氯化钴 （CoCl2） 诱导 2 种细胞 48 h 后的形态变化。分析 0、 10、 25、 50 及 100 mg/L CoCl2 处理 48 h 后缺氧诱导因子 （HIF） -1α蛋白表达变化， 筛选出 CoCl2诱导细胞缺氧的最适合浓度。MTT 实验检测不同时间点 （0 、 24 、 48 、 72 及 96 h） CoCl2诱导 2 种结直肠癌细胞的增殖情况， 筛选出 CoCl2诱导缺氧的最佳时间。最佳浓度和时间 条件下， 对 HCT116 和 HT-29 细胞分别进行缺氧 （缺氧组） 和常氧 （常氧组） 处理， Transwell 侵袭和划痕实验检测 2 组 2 种细胞的侵袭、 迁移情况； Western blot 实验和 RT-PCR 实验检测 2 组 HIF-1α、 E-cadherin 及 Vimentin 的蛋白及 mRNA 表达水平。结果 50 mg/L CoCl2作用 48 h 时 2 种细胞均出现明显的形态改变。2 组 HCT116、 HT-29 细胞的 HIF-1α蛋白表达水平随 CoCl2浓度增加均呈先增后减趋势， 50 mg/L 为最适宜浓度 （P < 0.05）。0~96 h 时 2 种细胞不 论有无缺氧， 细胞增殖能力均呈先增后减趋势 （P＜0.05）， 48 h 为最佳作用时间。HCT116 和 HT-29 细胞系中缺氧组 穿膜细胞数和细胞迁移率均明显高于常氧组 （P＜0.05）。HCT116、 HT-29 细胞系中缺氧组 HIF-1α、 Vimentin 的蛋白 和 mRNA 表达水平均高于常氧组， 而 E-cadherin 的蛋白和 mRNA 表达水平低于常氧组（均 P＜0.05）。结论 缺氧 能诱导不同分化程度结直肠癌细胞均发生 EMT， 并能增强 2 种结直肠癌细胞的侵袭、 迁移能力。

关键词: 缺氧诱导因子 1, α亚基, 结直肠肿瘤, 上皮间质转化, 细胞增殖, 肿瘤侵润, 细胞运动

Abstract: Abstract:Objective To explore whether hypoxia could promote epithelial-mesenchymal transition (EMT) in various differentiated colorectal cancer cells, and analyse the effect of hypoxia on invasion and migration of colorectal cancer cells. Methods HCT116 (poorly differentiated) and HT-29 (highly differentiated) colorectal adenocarcinoma cells were selected respectively. The morphological changes of two cell lines were observed after 0， 10， 25， 50， 100 and 150 mg/L cobalt chloride (CoCl2) treatment for 48 h. The expression of hypoxia-inducible factor-1α (HIF-1α) protein was analysed after 0， 10， 25， 50， 100 and 150 mg/L CoCl2 treatment for 48 h. An optimal concentration of CoCl2 was then selected. Methylthiazolyl tetrazolium (MTT) assay was used to detect the proliferation of two kinds of colorectal cancer cells induced by CoCl 2 at different time points (0, 24, 48, 72 and 96 h), and to select an optimal time. Under the optimal concentration and time conditions, the HCT116 and HT- 29 cells were processed by hypoxia (hypoxia group) and normoxia (normoxic group). Transwell invasion assay and Wound healing assay were used to detect cell invasion and migration in two groups. Western blot assay and RT-PCR were used to detect protein and mRNA expression levels of HIF-1α, E-cadherin and Vimentin in two groups. Results Two kinds of cells showed obvious morphological changes after 50 mg/L CoCl2 treatment for 48 h. HIF- 1α protein level first increased and then decreased in two groups of cells with the increased concentration of CoCl2, and 50mg/L CoCl2 was the optimal concentration (P＜0.05). The cell proliferation showed a tendency to decrease after the increase in both kinds of cells with or without hypoxia for 0-96 h (P＜0.05), and 48 h was the optimal time. The transmembrane number and cell migration rate were significantly more in hypoxia group than those of normoxic group (P＜0.05). The protein and mRNA levels of HIF- 1α and Vimentin were significantly higher in hypoxia group than those of normoxic group in HCT116 and HT-29 cell lines (P＜0.05). E-cadherin protein and mRNA levels were significantly lower in hypoxia group than those of normoxic group (P＜0.05). Conclusion Hypoxia can promote EMT in different differentiated colorectal cancer cells, and can enhance invasion and migration of two kinds of colorectal cancer cells.

Key words: hypoxia-inducible factor1,α subunit, colorectal neoplasms, epithelial-mesenchymal transition, cell prolifer? ation, neoplasm invasion, cell movement