Tspan8基因敲除联合安罗替尼对结肠癌SW480细胞增殖、迁移、侵袭和凋亡的影响

目的 探讨Tspan8基因敲除联合安罗替尼对结肠癌SW480细胞增殖、迁移、侵袭和凋亡的影响。方法 采用CRISPR/Cas9技术构建质粒并敲除SW480细胞的Tspan8基因，Western blot法检测敲除效果。采用MTT法计算安罗替尼的半数抑制浓度（IC₅₀）。实验分为对照组、安罗替尼组、Tspan8敲除组和联合组。采用细胞增殖实验、克隆形成实验、划痕实验、Transwell小室法和流式细胞术检测各组细胞的增殖、迁移、侵袭和凋亡情况；Western blot法检测安罗替尼对SW480细胞中Tspan8表达水平的影响。结果 在Tspan8敲除组中，SW480-KO-Ⅲ细胞的敲除效率最高，用于后续实验。不同浓度的安罗替尼在不同作用时间均能抑制SW480细胞的增殖（P<0.01），且呈浓度依赖性和时间依赖性（P<0.01），根据IC50选择14 μmol/L为后续实验浓度。与对照组相比，安罗替尼组、Tspan8敲除组和联合组的细胞增殖、迁移及侵袭能力显著降低，细胞凋亡水平明显提高（P<0.05），且联合组的上述变化较安罗替尼组或Tspan8敲除组更为显著（P<0.05）。与对照组相比，安罗替尼组SW480细胞的Tspan8表达水平明显下降（P<0.01）。结论 Tspan8基因敲除联合安罗替尼能协同抑制SW480细胞增殖、迁移、侵袭，并促进其凋亡。

Effects of Tspan8 Gene Knockout Combined with Anlotinib on Proliferation,Migration, Invasion and Apoptosis of Colon Cancer SW480 Cells

Objective To investigate the effects of Tspan8 gene knockout combined with anlotinib on the proliferation, migration, invasion, and apoptosis of colon cancer SW480 cells. Methods The plasmid was constructed by CRISPR/Cas9 technique, and Tspan8 gene was knocked out in SW480 cells. The knockout effect was detected by Western blot. The IC50 of anlotinib was calculated by MTT assay. The experiment was divided into control group, anlotinib group, Tspan8 knockout group, and combined group. Cell proliferation, migration, invasion, and apoptosis were detected by cell proliferation assay, clonal formation assay, scratch assay, Transwell chamber assay, and flow cytometry. Western blot was used to detect the effect of anlotinib on Tspan8 expression in SW480 cells. Results SW480-KO-Ⅲ cells had the highest knockout efficiency in the Tspan8 knockout group. They were used in subsequent experiments. Different concentrations of anlotinib could inhibit the proliferation of SW480 cells at different times (P<0.01) in a concentration-dependent and time-dependent manner (P<0.01). According to IC₅₀, 14 μmol/L was selected as the subsequent experimental concentration. Compared with those in the control group, the cell proliferation, migration, and invasion abilities in the anlotinib group, Tspan8 knockout group, and combined group were significantly decreased, and the cell apoptosis level was significantly increased (P<0.05). The above changes in the combined group were more significant than those in the anlotinib group or Tspan8 knockout group (P<0.05). Compared with that in the control group, the expression level of Tspan8 in SW480 cells in the anlotinib group was significantly decreased (P<0.01). Conclusion Tspan8 gene knockout combined with anlotinib can synergistically inhibit the proliferation, migration and invasion of SW480 cells. This combination can also promote the apoptosis of these cells.