白细胞介素17通过促进上皮间质转化调控胃癌细胞迁移侵袭能力

Effect of interleukin-17 in the migration and invasion of gastric cancer cell via regulating epithelialmesenchymal transition

    摘要
  • 摘要: 目的:探讨IL-17对胃癌细胞上皮间质转化(EMT)和侵袭迁移的影响及其可能作用机制。
    方法:(1)取对数生长期的胃癌细胞株MGC-803，分别运用浓度为0、1 ng/mL、10 ng/mL、100 ng/mL、 1 μg/mL IL-17干预48 h，观察细胞形态学变化。取细胞形态变化最为明显的浓度作为后续实验最适浓度。浓度为100 ng/mL的IL-17干预胃癌细胞48h，设为实验组；加入等量PBS干预胃癌细胞48 h，设为对照组。(2)RTPCR检测两组胃癌细胞中钙粘附蛋白E(Ecadherin)、波形蛋白(Vimentin)的RNA表达水平。(3)Western blot检测两组胃癌细胞中Ecadherin和Vimentin的相对蛋白表达量。(4)划痕实验和 Transwell实验检测两组胃癌细胞的侵袭和迁移能力。正态分布的计量资料采用±s表示，两组比较采用 t检验。
    结果:(1)胃癌细胞EMT形态变化：不同浓度IL-17处理MGC-803胃癌细胞48 h后，细胞形态发生显著改变。主要是细胞由多角紧密连接状态逐渐向连接松散，梭形形态转变，细胞粘附能力明显下降，且随着IL-17浓度从0增加至100 ng/mL时，细胞形态改变逐渐明显；当浓度达到100 ng/mL时，细胞形态改变最明显；但当IL-17浓度继续增加至1 μg/mL时，细胞形态改变不再显著，部分细胞出现死亡，漂浮现象。(2)RTPCR检测结果：实验组和对照组胃癌细胞中Ecadherin mRNA相对表达量分别为0.45±0.13和1.06±0.23；Vimentin mRNA相对表达量分别为2.39±0.55和1.23±0.41，上述指标两组比较，差异均有统计学意义(t=3.811，2.923，P<0.05)。(3)Western blot 检测结果：实验组和对照组胃癌细胞中Ecadherin蛋白相对表达量分别为0.86±0.17和1.56±0.29；Vimentin蛋白相对表达量分别为1.01±0.12和0.56± 0.17，上述指标两组比较，差异均有统计学意义(t=3.551，3.601，P<0.05)。(4)划痕实验结果显示：划痕36 h后，实验组和对照组胃癌细胞均发生了迁移，其划痕宽度分别为(0.76±0.13)mm和(0.40±0.15)mm，两组比较，差异有统计学意义(t=3.095，P<0.05)。Transwell法检测结果显示：实验组和对照组穿膜细胞数计数为(159±28)个和(94±18)个，两组比较，差异有统计学意义(t=3.307，P<0.05)。
    结论:IL-17通过促进胃癌细胞发生EMT转变，进而增强胃癌细胞迁移侵袭能力。

     

    Abstract: Objective:To investigate the effect of interleukin-17 (IL-17) in the gastric cancer cell migration and invasion via regulating epithelialmesenchymal transition (EMT) and its potential function.
    Methods (1) Human gastric cancer cell MGC-803 lines in the logarithmic growth phase were stimulated by 0, 1 ng/mL, 10 ng/mL,100 ng/mL and 1μg/mL of IL-17 for 48 hours, and the phenotypic changes were observed. The concentration of IL-17 was selected for followon experiments based on the most obvious phenotypic changes. Gastric cancer cell MGC-803 which were stimulated by 100 ng/mL of IL-17 and PBS for 48 hours were allocated into the experimental group and control group, respectively. (2) The expressions of Ecadherin and Vimentin mRNA in gastric cancer cells were assayed through realtime PCR (RTPCR). (3) The relative expressions of Ecadherin and Vimentin proteins in gastric cancer cells were assayed by the Western blot. (4) The scratch test and Transwell detection were also utilized to study the migration and invasion of gastric cancer cell MGC-803 in vitro. Measurement data with normal distribution were presented as ±s and comparison between groups was analyzed using the t test.
    Results:(1) There were significant phenotypic changes in the gastric cancer cell after the different concentration of IL-17 stimulated gastric cancer cell MGC-803 for 48 hours. Cells were changed from polygonal and tight junction to spindle and loosely junction with a deterioration of cell adhesion. Cell phenotypes were gradually changed as the concentration of IL-17 was changed from 0 to 100 ng/mL. Phenotypic changes were the most obvious when 100 ng/mL of IL-17 was used, but these were nonsignificant as the concentration of IL-17 increased to 1 μg/mL with the death and floating of some cells. (2) The relative expressions of Ecadherin mRNA and Vimentin mRNA in RTPCR were 0.45±0.13 and 1.06±0.23 in the experimental group and 2.39±0.55 and 1.23±0.41 in the control group, respectively, with significant differences (t=3.811, 2.923, P<0.05). (3) The results of Western blot showed the relative expressions of Ecadherin and Vimentin proteins were 0.86±0.17 and 1.56± 0.29 in the experimental group and 1.01±0.12 and 0.56±0.17 in the control group, respectively, with significant differences (t=3.551, 3.601, P<0.05). (4) Cell migration in the 2 groups were detected by the scratch test at 36 hours after scratch test, and the width of scratch in the experimental and control groups were (0.76±0.13)mm and (0.40±0.15)mm, showing a significant difference (t=3.095, P<0.05). Transwell detection showed number of transmembrane cell in the experimental and control groups were 159±28 and 94±18, respectively, with a significant difference (t=3.307, P<0.05).
    Conclusion:IL-17 can promote the migration and invasion of gastric cancer cells via stimulating alteration of EMT.

     

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