Effect of AK4 on Proliferation and Migration of Intrahepatic Bile Duct Carcinoma Cell HUCCT1
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摘要:
目的 探究AK4对肝内胆管癌细胞HUCCT1增殖、迁移能力的影响。 方法 采用小干扰RNA（siRNA）技术靶向沉默肝内胆管癌细胞HUCCT1中AK4的表达，采用免疫印迹法检测沉默效果以及筛选siRNA-AK4，通过EdU实验和细胞划痕实验检测该胆管癌细胞增殖、迁移能力。 结果 通过免疫印迹法检测出siRNA-AK4-3沉默效果最好，EdU实验显示沉默AK4后，细胞增殖能力下降（P < 0.05），细胞划痕实验显示沉默AK4后，细胞迁移能力下降（P < 0.01）。 结论 沉默肝内胆管癌细胞HUCCT1中AK4的表达后，其增殖、迁移能力受到抑制。 Abstract:Objective To investigate the effects of AK4 on the proliferation and migration of intrahepatic bile duct carcinoma cells HUCCT1. Methods Small interfering RNA （siRNA） technology was used to target the silencing of AK4 expression in HUCCT1 of intrahepatic bile duct cancer cells. Western blot was used to detect the transfection efficiency of siRNA and screen siRNA-AK4. The proliferation and migration ability of the bile duct cancer cells were detected by EdU assay and cell scar assay. Results EdU experiment showed that cell proliferation decreased after AK4 silencing （P < 0.05）, the cell migration ability was decreased after AK4 silencing （P < 0.01）. Conclusion AK4 can enhance the proliferation and migration of HUCCT1 in intrahepatic bile duct carcinoma cells. -
Key words:
- Intrahepatic cholangiocarcinoma /
- AK4 /
- Proliferation /
- Migration
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图 2 各组细胞中AK4蛋白的相对表达量
siRNA-GAPDH组为阳性对照，取GAPDH/AK4。各组分别与siRNA-NC组比较，除CON组无统计学差异其余各组均有统计学差异。**P < 0.01，***P < 0.005，****P < 0.001。
Figure 2. The relative expression level of AK4 protein in each group
图 3 EdU检测siRNA-NC与siRNA-AK4-3细胞的增殖情况（×200）
Figure 3. The proliferation of siRNA-NC and siRNA-AK4-3 cells was detected by EdU （×200）
图 4 siRNA-NC组与siRNA-AK4-3的细胞增殖比率
*P < 0.05。
Figure 4. The proliferation rate of siRNA-NC group and siRNA-AK4-3
图 5 siRNA-NC与siRNA-AK4-3细胞划痕后面积变化，图中红色边缘为Image J软件勾勒
Figure 5. The area changes of siRNA-NC and siRNA-AK4-3 cells after scratches，and the red edge in the figure is outlined by Image J software
图 6 siRNA-NC组与siRNA-AK4-3组细胞划痕后面积占视野总面积比的变化
*P < 0.05，**P < 0.01。
Figure 6. In the siRNA-NC group and siRNA-AK4-3 group，ns represented no difference in the ratio of cell area to total field area after scratches
表 1 si-RNA序列
Table 1. Sequence of si-RNA built by siRNA technology
组别 序列 Antisense Negative control 5′-UUCUCCGAACGUGUCACGUTT-3′ 5′-ACGUGACACGUUCGGAGAATT-3′ GAPDH Positive control 5′-UGACCUCAACUACAUGGUUTT-3′ 5′-AACCAUGUAGUUGAGGUCATT-3′ siRNA-AK4-1 5′-GCGGAAGGGUAUAUAACCUTT-3′ 5′-AGGUUAUAUACCCUUCCGCCT-3′ siRNA-AK4-2 5′-CAGGCUAAGACAGUACAAATT-3′ 5′-UUUGUACUGUCUUAGCCUGTT-3′ siRNA-AK4-3 5′-CACCUAUUCAGUCCAAAGATT-3′ 5′-UCUUUGGACUGAAUAGGUGTT-3′ 
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表 2 划痕面积比
Table 2. Scratch area ratio
项目 0 h 12 h 24 h 36 h siRNA-NC 0.39 ± 0.01 0.26 ± 0.017 0.12 ± 0.017 0.08 ± 0.026 siRNA-AK4-3 0.39 ± 0.005 0.33 ± 0.001 0.24 ± 0.024 0.24 ± 0.027
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