EIF5A1 Promotes Proliferation，Migration and Invasion of Intrahepatic Cholangiocarcinoma Cells Through Wnt Signaling Pathway
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摘要:
目的 研究EIF5A1在肝内胆管癌细胞株和人肝内胆管上皮中的表达及其对HUCCT1细胞增殖、迁移侵袭和Wnt/β-Catenin信号通路的影响。 方法 使用Western blot方法检测EIF5A1在肝内胆管癌细胞株和人肝内胆管上皮细胞中的基础表达量，采用瞬时转染siRNA的方法沉默肝内胆管癌细胞HUCCT1中EIF5A1的表达，实验分组为空白对照组（Con）、siRNA1组、siRNA2组。通过Western blot方法筛选沉默效果最佳的siRNA，CCK-8、EdU细胞增殖实验和Transwell实验检测沉默EIF5A1后对HUCCT1细胞增殖和迁移侵袭能力的影响，Western blot检测HUCCT1细胞中沉默EIF5A1后对Wnt/β-Catenin信号通路的影响。 结果 CCK-8、EdU细胞增殖实验结果显示沉默EIF5A1后，细胞增殖能力下降（P < 0.05），Transwell迁移侵袭实验结果显示沉默EIF5A1后抑制了HUCCT1细胞迁移及侵袭（P < 0.05），沉默EIF5A1后Wnt/β-Catenin信号通路中的β-Catenin、Cyclin D1、MMP-2和Survivin蛋白表达明显下降（P < 0.05）。 结论 EIF5A1可能通过Wnt/β-Catenin信号通路促进肝内胆管癌细胞增殖和迁移侵袭。 -
关键词:
- 肝内胆管癌 /
- EIF5A1 /
- Wnt/β-Catenin信号通路 /
- 增殖 /
- 迁移侵袭
Abstract:Objective To investigate the expression of EIF5A1 in intrahepatic cholangiocarcinoma cell lines and human hepatobiliary duct epithelia, and its effect on the proliferation, migration and invasion ability and Wnt/β-Catenin signaling pathway in HUCCT1 cells . Methods Western blot was used to detect the basal expression level of EIF5A1 in intrahepatic cholangiocarcinoma cell lines and human intrahepatic cholangiocarcinoma epithelial cells. Transient transfection of siRNA was used to silence the expression of EIF5A1 in intrahepatic cholangiocarcinoma cell HUCCT1. The experimental groups were divided into blank control group (Con), siRNA1 group, and siRNA2 group. The most effective siRNA was screened by Western blot. The effects of EIF5A1 silencing on the proliferation, migration and invasion ability of HUCCT1 cells were detected by CCK-8, EdU cell proliferation assay and Transwell assay. The effect of EIF5A1 silencing on the Wnt/β-Catenin signaling pathway in HUCCT1 cells was detected by Western blot. Results The results of CCK-8 and EdU cell proliferation experiments showed that the proliferation ability of HUCCT1 cells decreased after EIF5A1 silencing （P < 0.05）, and Transwell migration and invasion experiments showed that the migration and invasion ability of Hucct1 cells decreased after EIF5A1 silencing （P < 0.05）. Western blot analysis revealed decreased expression of β-Catenin, Cyclin D1, MMP-2 and Survivin in Wnt/β-Catenin signaling pathway after EIF5A1 silencing （P < 0.05）. Conclusion EIF5A1 may promote the proliferation, migration and invasion of intrahepatic bile duct cancer cells through Wnt/β-Catenin signaling pathway. -
图 1 EIF5A1在肝内胆管癌细胞株及肝内胆管上皮细胞中的基础表达量（$ \bar x \pm s $，n = 3）
A：EIF5A1在肝内胆管癌细胞株及肝内胆管上皮细胞中的免疫印迹图；B：EIF5A1蛋白相对表达量柱状图。*P < 0.05，**P < 0.001，***P < 0.0001。
Figure 1. The basal expression levels of EIF5A1 in intrahepatic cholangiocarcinoma cell lines and intrahepatic bile duct epithelial cells （$ \bar x \pm s $，n = 3）
图 2 不同siRNA 对HUCCT1细胞中EIF5A1的沉默效果（$ \bar x \pm s $，n = 3）
A：不同siRNA沉默HUCCT1细胞中EIF5A1表达的免疫印迹图；B：EIF5A1蛋白相对表达量柱状图；**P < 0.001。
Figure 2. Silencing effects of different siRNA on EIF5A1 in HUCCT1 cells（$ \bar x \pm s $，n = 3）
图 3 CCK-8实验显示Con组和siRNA2组细胞的增殖情况（$ \bar x \pm s $，n = 5）
*P < 0.05。
Figure 3. CCK-8 assay showing proliferation of cells in Con group and siRNA2 groups （$ \bar x \pm s $，n = 5）
图 4 EdU实验显示Con组和siRNA2组细胞的增殖率（$ \bar x \pm s $，n = 3）
A：siRNA2沉默EIF5A1后对HUCCT1细胞增殖的影响（×200）；B：沉默EIF5A1后细胞增殖率柱状图；*P < 0.05。
Figure 4. EdU assay showing the proliferation rate of cells in Con and siRNA2 groups（$ \bar x \pm s $，n = 3）
图 5 siRNA2沉默HUCCT1细胞中EIF5A1后对其迁移、侵袭能力的影响（$ \bar x \pm s $，n = 3）
A：siRNA2沉默EIF5A1后对HUCCT1细胞迁移的影响（×200）；B：迁移细胞柱状图；C：siRNA2沉默EIF5A1后对HUCCT1细胞侵袭的影响（×200）；D：侵袭细胞柱状图；*P < 0.05。
Figure 5. Effects of siRNA2 silencing EIF5A1 on its migration and invasion ability in HUCCT1 cells（$ \bar x \pm s $，n = 3）
图 6 沉默HUCCT1细胞中EIF5A1的表达对Wnt/β-Catenin信号通路的影响（$ \bar x \pm s $，n = 3）
A：沉默EIF5A1后 Wnt/β-Catenin信号通路关键蛋白免疫印迹图；B：蛋白相对表达量柱状图；*P < 0.05，**P < 0.001。
Figure 6. Effects of silencing EIF5A1 expression in HUCCT1 cells on the Wnt/β - Catenin signaling pathway（$ \bar x \pm s $，n = 3）
表 1 siRNA序列信息
Table 1. siRNA sequence information
siRNA 名称 序列（5'→3'） siRNA1 EIF5A1-si-F GUCGAGAUGUCUACUUCGATT EIF5A1-si-R UCGAAGUAGACAUCUCGACTT siRNA2 EIF5A1-si-F CACGUGAGUUGGAAUCGAATT EIF5A1-si-R UUCGAUUCCAACUCACGUGTT 
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