慢病毒稳定下调ADP核糖基化因子的GTP酶激活蛋白1表达对肺腺癌细胞增殖和侵袭的影响  

The effects of slow virus-induced downregulation of ArfGAP with SH3 domain,ankyrin repeat and PH domain 1 expression on the proliferation and invasion of lung adenocarcinoma cells

作　　者：陈晓铭 杨志昌 谷倬宇 赵阳 杨洋[1] Chen Xiaoming;Yang Zhichang;Gu Zhuoyu;Zhao Yang;Yang Yang(Department of Thoracic Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院胸外科,郑州450052

出　　处：《中华实验外科杂志》2023年第10期2039-2042,共4页Chinese Journal of Experimental Surgery

基　　金：河南省高等学校重点科研项目(22A320072);河南省中青年卫生健康科技创新人才培养(领军人才)项目(YXKC2021016);河南省医学科技攻关计划省部共建项目(重点项目)(SBGJ202102122)。

摘　　要：目的探讨慢病毒稳定下调腺苷二磷酸核糖基化因子的三磷酸鸟苷酶激活蛋白1(ASAP1)表达对肺腺癌细胞增殖和侵袭的影响。方法收集郑州大学第一附属医院病理科2022年8月1日至2023年5月1日手术切除肺腺癌及配对癌旁组织各5例,免疫组织化学(IHC)检测ASAP1在人肺腺癌组织中的表达,结合在线数据库(KM-plotter、HPA、UALCAN)分析ASAP1表达与肺腺癌患者预后的关系。在体外用ASAP1阴性对照慢病毒(NC)和ASAP1敲低短发夹RNA慢病毒(ASAP1 KD)感染A549和HCC4006细胞,构建稳定下调ASAP1表达的肺腺癌细胞系。蛋白印迹(WB)检测ASAP1蛋白下调效率,通过平板克隆实验、细胞活力检测试剂盒(CCK-8)、迁移实验、Transwell侵袭实验、蛋白印迹检测下调ASAP1对细胞增殖侵袭的影响和对上皮-间充质转化(EMT)相关蛋白表达的影响,两组间比较采用独立样本t检验。结果ASAP1在人肺腺癌组织中的表达远高于癌旁正常组织(209.70±9.02比48.33±2.89,t=29.51,P<0.01),且多个数据库显示ASAP1的表达与患者不良预后明显相关[KM-plotter数据库(P<0.0001)、HPA数据库(P<0.001)、UALCAN数据库(P<0.01)];体外实验结果显示,较NC组,ASAP1 KD组的ASAP1蛋白表达明显下调(A549细胞株:0.99±0.03比0.36±0.01,t=27.39,P<0.01;HCC40006细胞株:1.02±0.07比0.27±0.07,t=11.08,P<0.01),细胞形成的克隆数量明显减少(A549细胞株:233.70±5.51比33.67±10.02,t=69.28,P<0.01;HCC4006细胞株:333.3±30.55比50.33±5.508,t=14.17,P<0.01),细胞在450nm波长处的吸光度值降低(A549细胞株:1.18±0.06比0.64±0.11,t=8.20,P<0.05;HCC4006细胞株:0.94±0.04比0.57±0.09,t=4.79,P<0.05)。此外,ASAP1 KD组穿过小室成功迁移的细胞数量明显减少(A549细胞株:137.00±8.54比19.67±5.03,t=32.00,P<0.01;HCC4006细胞株:42.67±3.51比12.33±2.51,t=45.50,P<0.01),穿过基质胶成功侵袭的细胞数量明显减少A549细胞株:207.70±9.07比(41.33±4.73,t=49.17,P<0.01;HCC4006细胞株:35.00±5.00比6.67±1.53,Objective Investigations were conducted on the impact of downregulating ArfGAP with SH3 domain,ankyrin repeat and PH domain 1(ASAP1)expression on the proliferation and invasion of lung adenocarcinoma cells in the context of stable lentivirus-mediated delivery of slow viral particles.Methods Collect 5 cases of lung adenocarcinoma surgical resections and paired adjacent cancer tissue from the Pathology Department of the First Affiliated Hospital of Zhengzhou University between August 1,2022,and May 1,2023.Conduct immunohistochemistry(IHC)analysis to examine ASAP1 expression in the lung adenocarcinoma tissue.Analyze its correlation with patient prognosis using online databases like KM-plotter,HPA,and UALCAN.In vitro experiments involved infecting A549 and HCC4006 cells with ASAP1 knockdown lentiviral particles(ASAP1 KD)and negative control lentiviral particles(NC)to establish lung adenocarcinoma cell lines with stable downregulated ASAP1 expression.The efficiency of ASAP1 knockdown was assessed using Western blotting analysis.The effects of downregulated ASAP1 on cell proliferation,invasion,and epithelial-mesenchymal transition(EMT)-related protein expression were evaluated using colony formation assays,cell viability assays(CCK-8),migration assays,Transwell invasion assays,and Western blotting analysis.The comparison between two groups is conducted using an independent samples t-test.Results ASAP1 expression in human lung adenocarcinoma tissue was significantly higher compared to adjacent normal tissue(209.70±9.02 vs.48.33±2.89,t=29.51,P<0.01).Multiple databases indicated a correlation between ASAP1 expression and unfavorable prognosis in patients[KM-plotter database(P<0.0001),HPA database(P<0.001),UALCAN database(P<0.01)].In vitro experiments revealed that ASAP1 protein expression was significantly downregulated in the ASAP1 KD group compared to the NC group(A549 cells:0.99±0.03 vs.0.36±0.01,t=27.39,P<0.01;HCC40006 cells:1.02±0.07 vs.0.27±0.07,t=11.08,P<0.01).The number of cell clones formed significantly de

关 键 词：肺腺癌 腺苷二磷酸核糖基化因子的三磷酸鸟苷酶激活蛋白 增殖 侵袭 上皮-间充质转化 

分 类 号：R734.2[医药卫生—肿瘤]