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目的在皮肤鳞癌细胞(A431)中下调VEGF mRNA和VEGF蛋白的表达,观察其对A431细胞增殖、迁移和黏附的影响。方法构建psVEGF-shRNA真核表达质粒(psilencer-VEGF1-shRNA,VEGF-s1;psilencer-VEGF2-shR-NA,VEGF-s2)干预A431细胞,同时构建含随机靶序列的阴性对照表达质粒(psilencer-Target-off-shRNA,T-off)。RT-QPCR,Western blot和ELISA检测细胞内VEGFmRNA和蛋白表达;CCK-8法检测细胞活性;流式细胞仪检测细胞周期百分比与细胞凋亡;划痕试验和Transwell小室试验检测细胞二维、三维空间的迁移能力;FN黏附试验检测细胞黏附潜能。结果 A431细胞转染psVEGF-shRNA后活性降低,细胞周期发生阻滞,与对照组相比细胞比率显著增加,在G1期明显降低,在S期伴细胞调亡增加。细胞内VEGFmRNA和VEGF蛋白表达下降,细胞迁移和黏附潜能力均受到抑制(P<0.05)。结论 VEGF是人皮肤鳞癌A431细胞生长相关的重要基因,干扰VEGF基因能有效抑制A431细胞的增殖、迁移和黏附。
OBJECTIVE: To downregulate the expression of VEGF mRNA and VEGF protein in the skin squamous cell carcinoma (A431) cells and observe the effect on the proliferation, migration and adhesion of A431 cells. Methods The A431 cells were induced by psilencer-VEGF1-shRNA (psilencer-VEGF1-shRNA, psilencer-VEGF2-shR-NA and VEGF-s2) and a negative control expression vector (psilencer- Target-off-shRNA, T-off). RT-qPCR, Western blot and ELISA were used to detect the expression of VEGF mRNA and protein. CCK-8 method was used to detect the cell viability. Flow cytometry was used to detect cell cycle percentage and apoptosis. Scratch assay and Transwell chamber assay were used to detect the cell 2D and 3D Space migration ability; FN adhesion test to detect cell adhesion potential. Results After transfection with psVEGF-shRNA, A431 cells showed decreased activity and cell cycle arrest. Compared with the control group, the percentage of cells increased significantly, decreased in G1 phase and increased in S phase. The expression of VEGFmRNA and VEGF in the cells decreased, the ability of cell migration and adhesion was inhibited (P <0.05). Conclusion VEGF is an important gene related to the growth of human skin squamous cell carcinoma A431 cells. Interfering with VEGF gene can effectively inhibit the proliferation, migration and adhesion of A431 cells.