The Effect of HIF-1α on Invasion and Metastasis of Gallbladder Cancer by Regulating the Epithelial-Mesenchymal Transformation
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摘要:
目的 探讨HIF-1α对胆囊癌细胞迁移侵袭的影响及其相关调控机制。 方法 构建GBC-SD细胞，使用慢病毒转染，分别得到实验组（HIF-1α shRNA）、阴性对照组（空载慢病毒转染）和空白对照组（未处理）。蛋白质免疫印迹法检测E-Cadherin、N-Cadherin蛋白的表达水平，GBC-SD细胞的迁移能力用细胞划痕实验检测，GBC-SD细胞的侵袭能力用Transwell 侵袭实验检测。 结果 与对照组相比，实验组E-Cadherin蛋白的表达水平明显升高（P < 0.001），N-Cadherin蛋白的表达水平明显降低（P < 0.001），对照组之间E-Cadherin、N-Cadherin蛋白的表达水平无明显差异（P > 0.05）。实验组细胞划痕愈合度较对照组低（P < 0.001）、穿模个数较对照组少（P < 0.001），而对照组之间细胞划痕愈合度、穿模个数无明显差异（P > 0.05）。 结论 靶向敲低HIF-1α可以降低GBC-SD细胞的迁移侵袭能力，其作用机制可能与调控上皮-间充质转化有关。 Abstract:Objective To explore the effect of HIF-1α on the migration and invasion of gallbladder cancer cells and its related regulatory mechanism. Methods GBC-SD cells were constructed and transfected with lentivirus, and the experimental group （HIF-1α shRNA）, negative control group （empty lentivirus transfection） and blank control group （untreated） were obtained respectively. The expression levels of E-Cadherin, and N-Cadherin proteins were detected by Western blotting assay, Cell scratch assay was ued to detect the migratory ability of GBC-SD cells, and Transwell invasion assay was uesd to detect the invasive ability of GBC-SD cells. Results Compared with the control group, the expression level of E-Cadherin protein in the experimental group was significantly increased （P < 0.001）, while the expression level of N-Cadherin protein was significantly decreased （P < 0.001）, and there was no remarkable difference in the expression level of E-Cadherin and N-Cadherin proteins between the control groups （P both>0.05）. In the experimental group, the cell scratch healing degree was inferior to the control group （P < 0.001） , and the number of penetrating molds was also inferior to the control group （P < 0.001）, while there was no obvious otherness in the cell scratch healing degree and the number of penetrating molds between the control groups （P > 0.05）. Conclusion Targeted knockdown of HIF-1α can reduce the migration and invasion ability of gallbladder cancer cells, and its mechanism of action may be related to the regulation of epithelial mesenchymal transition. -
图 1 各组GBC-SD细胞E-Cadherin、N-Cadherin蛋白的表达水平
A：各组E-Cadherin和N-Cadherin蛋白表达电泳图；B：E-Cadherin蛋白表达分析；C:N-Cadherin蛋白表达分析；ns为P > 0.05，***P < 0.001。
Figure 1. Expression levels of E-Cadherin and N-Cadherin proteins in GBC-SD cells of each group
图 2 各组GBC-SD细胞的迁移能力
A：24 h、48 h后3组细胞划痕愈合情况（×40）；B：24 h、48 h后3组细胞划痕愈合率；ns为P > 0.05，***P < 0.001。
Figure 2. Migratory capacity of GBC-SD cells in each group
图 3 各组GBC-SD细胞的侵袭能力
A：结晶紫染色后3组细胞穿模情况（×100）；B：3组细胞穿模个数；ns 为P > 0.05，***P < 0.001。
Figure 3. Assay of invasive ability of each group of GBC-SD cells
表 1 抗体信息
Table 1. Antibody information
抗体名称 厂家 货号 稀释浓度 种属 GAPDH PROTEINTECH 10068-1-AP 1∶5000 Mouse HIF1α Affinity Biosciences AF1009 1∶5000 Rabbit E-Cadherin Affinity Biosciences AF0131 1∶5000 Rabbit N-cadherin Affinity Biosciences AF5239 1∶5000 Rabbit Anti-Rabbit IgG （H+L） Antibody，Peroxidase-Labeled KPL 074-1506 1∶5000 Anti-Mouse IgA + IgG + IgM （H+L） Antibody，Human Serum Adsorbed and Peroxidase-Labeled KPL 074-1807 1∶5000 
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表 2 各组细胞愈合度和穿模细胞数（$ \bar x \pm s$）
Table 2. Cell healing degree and number of perforated cells in each group （$ \bar x \pm s $）
组别 48 h划痕愈合度（%） 穿模细胞数（个） Control组 69.96$ \pm $0.47 215.00$ \pm $2.94 sh-NC组 69.25$ \pm $0.86 215.33$ \pm $2.05 sh-HIF1α组 44.51$ \pm $0.93ab 121.67$ \pm $1.69ab χ2/F 18.00 1108.18 P <0.001* <0.001* 与sh-NC组比较，aP < 0.001；与Control组比较，bP < 0.001；*P < 0.05。
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