靶向VEGF的shRNA对皮肤鳞癌A431细胞的作用

 目的 构建靶向VEGF的shRNA（psilencer-VEGF1-shRNA，VEGF-s1；psilencer-VEGF2-shRNA，VEGF-s2）干预皮肤鳞癌细胞（A431）的一系列生物学行为，探讨VEGF-s1和VEGF-s2干预作用的意义。方法 构建psVEGF-shRNA真核表达质粒干预体外培养的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-shrank，T-off)。RT-QPCR,western blot 和ELISA检测细胞内VEGFmRNA和蛋白表达水平的变化；CCK-8法检测细胞活性；流式细胞仪检测癌细胞增殖周期细胞百分比与细胞凋亡；划痕试验和Transwell小室试验别检测癌细胞二维和三维空间的迁移能力；FN黏附试验检测癌细胞的黏附潜能。结果 A431细胞转染psVEGF-shrank后活性降低，细胞周期发生阻滞，与对照组相比细胞在G1期比率显著增加(P<0.05)，在S期比率明显降低，细胞调亡增加(均P<0.05)，细胞内VEGFmRNA和蛋白表达水平下降，细胞迁移和黏附潜能均受到抑制。结论 VEGF是人皮肤鳞癌细胞生长相关的重要基因，干扰VEGF基因能有效抑制人皮肤鳞癌细胞的生物学行为。

Effect of shRNA targeting VEGF on human skin squamous carcinoma cell line A431

【Abstract】Objective The eukaryotic expression plasmids targeting VEGF (psilencer-VEGF1-shRNA，VEGF-s1；psilencer-VEGF2-shRNA，VEGF-s2)were constructed and transfected these plasmids to the human skin squamous cell carcinoma cell line (A431) to observe its affect to a series of biological characteristics of A431 cells for exploring the significance of intervention by VEGF-s1 and VEGF-s2. Method: VEGF-s1,VEGF-s2 and random target sequence of the negative control plasmid (psilencer-Target-off-shRNA，T-off)were constructed, simultaneously. and transfected these plasmids to A431 by Tm2000. The expression of VEGF mRNA and protein were detected by RT-QPCR, western blot and ELISA; the vitality of A431 cells was examined by CCK-8 assay; the percentage of Cell Proliferative Cycle and apoptosis of A431 were tested by flow cytometry; the migration capacity of A431 in two-dimensional and three- dimensional space were inspected by wound healing effect and Transwell assay,respectively; the adhesion potential of A431 was detected by FN adhesion test. Results: After VEGF-s1 or VEGF-s2 treatment, cytoactivity declined, cell cycle arrested, G1 cell proportion increased sharply, S cell proportion decreased obviously, apoptosis increased, the expression of VEGF mRNA and protein decreased significantly, the migration and adhesion of A431 cells was suppressed significantly, as compare with the control group and untreated cells(every ,p<0.05). Conclusion: VEGF is an important growth-related gene of human skin squamous cell carcinoma, silencing VEGF could effectively inhibit the biological behavior of A431 cells.