PAK1基因与皮肤鳞状细胞癌迁移侵袭的研究

目的探讨短发夹RNA(shRNA)沉默p21活化激酶1(p21-activatedkinase1,PAKl)基因对皮肤鳞癌A431细胞系迁移及侵袭能力的影响。方法合成PAKl特异性shRNA(PAK1-shRNA)和通用无义shRNA序列(NCshRNA),转染A431细胞;随机将A431细胞分为空白对照组A431组、阴性对照组NC-shRNA-A431组及转染组pG1.1-PAK1-shRNA-A431组,转染细胞后,通过RT-PCR、WB检测比较空白对照组、阴性对照组及转染组细胞PAK1 mRNA、蛋白表达水平的差异;Transwell法检测转染后细胞迁移及侵袭能力的变化。结果成功转染A431细胞后,RT-PCR及WB检测结果表明,转染组pG1.1-PAK1-shRNA-A431较空白对照组A431组、阴性对照组NC-shRNA-A431组的PAK1mRNA水平及蛋白表达水平表达均明显下调;Transwell检测细胞迁移及侵袭能力结果显示,pG1.1-PAK1-shRNA-A431组细胞迁移及侵袭细胞数量较空白对照组A431组、阴性对照组NC-shRNA-A431组明显减少。结论shRNA干扰PAK1基因能使A431中的PAK1表达量明显下调,并可明显降低A431细胞的迁移及侵袭能力。提示PAK1可能作为一个潜在的无创性分子指标来评价鳞状细胞癌的侵袭能力。

Study between PAK1 gene and migration and invasion of skin squamous cell carcinoma

Objective To investigate the effects of short hairpin RNA targeting p2 l activated kinases l(PAKl)gene on migration and invasion of cutaneous squamous carcinoma cell line A431. Methods Synthesized shRNA targeting PAKl(PAKl-shNRA) and negative control shRNA(NC-shRNA) were transfected into A431 cells. The A431 cells were divided into three groups: blank control group(A431 group), negative control group(NC-shRNA-A431 group) and experimental group(pG1.1-PAK1-shRNA-A431 group). Real time PCR and Western blot analysis were performed to detect mRNA and protein expressions of PAK1 in these cells after transfection. Transwell assay was conducted to evaluate migratory and invasive activities of cells. Results The expressions of PAK1 mRNA and protein in pG1.1-PAK1-shRNAA431 cells were significantly decreased compared with those in the NC-shRNA-A431 and A431 cells. Transwell migration and invasion assay revealed that the number of A431 cells crossing the Transwell chamber was significantly lower in the transfection group than in the control group and negative group respectively. Conclusions PAK1-shRNA can obviously decrease PAKl expression and inhibit the ability of migration and invasion in A431 cells;PAK1 may be a potential noninvasive molecular marker to evaluate the invasive ability of squamous cell carcinoma.