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Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion

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Author:: No author available

Journal Title:: Acta Physiologica Sinica

Issue:: 2

DOI:: 10.13294/j.aps.2018.0032

Key Word:: UCHL1;CRISPR-CAS9;非小细胞肺癌;耐药性;迁移

Abstract：本研究旨在探索泛素羧基末端水解酶L1 (ubiquitin C-terminal hydrolase-L1,UCHLl)对非小细胞肺癌细胞系A549细胞的作用.用CRISPR-CAS9基因编辑技术构建UCHL1基因敲除的A549细胞株,用RT-PCR和Western blot检测A549细胞中UCHL1基因敲除情况,用CCK-8检测细胞增殖能力的变化,用流式细胞仪检测细胞周期的变化,用CCK-8检测A549细胞对顺铂药物敏感性的改变,用划痕与Transwell实验检测A549细胞迁移能力的变化,用Western blot检测与A549细胞迁移有关的蛋白表达变化.结果显示,使用CRISPR-CAS9技术构建的基因移码突变导致A549细胞株UCHL1 mRNA和蛋白缺失,UCHL1基因功能缺失后A549细胞增殖和各细胞周期比例没有明显变化,但对顺铂的药物敏感性降低,迁移能力下降,Erk 1/2蛋白磷酸化水平升高.以上结果提示,UCHL1基因功能缺失可导致A549细胞顺铂耐药性提高,细胞迁移能力降低,其机制可能涉及Erk1/2信号通路的激活.

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