氯化钴通过c-Raf/MAPK/NF-κB通路对人非小细胞肺癌细胞增殖、侵袭及迁移影响  

Effect of cobalt chloride on proliferation, invasion and migration of human non-small cell lung cancer cells via c-Raf/MAPK/NF-κB pathway

作　　者：牛海文 曹国磊 崔浩波 何丽丽 曹嘉芮 李晓晗 罗琴 NIU Hai-wen;CAO Guo-lei;CUI Hao-bo;HE Li-li;CAO Jia-rui;LI Xiao-han;LUO Qin(Department of Respiratory Neurology,Third Clinical College of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第三临床医学院呼吸神经内科,新疆乌鲁木齐830011

出　　处：《中华肿瘤防治杂志》2022年第7期475-480,500,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81760014);天山青年计划(2018Q048);新疆自治区科技支疆计划(2019E0281)。

摘　　要：目的探讨体外缺氧微环境对c-Raf/MAPK/NF-κB通路的影响,并观察不同浓度氯化钴(CoCl_(2))及作用不同时间对人非小细胞肺癌(NSCLC)细胞增殖、侵袭及迁移的影响。方法采用CoCl_(2)建立人NSCLC细胞系NCI-H1650和A549细胞体外化学乏氧模型,以作用浓度0μmol/L为对照组,作用不同浓度为实验组。通过不同浓度(100、200、300、400和500μmol/L)的CoCl_(2)分别作用NCI-H1650和A549细胞12 h,CCK-8法检测其存活率。根据半数抑制浓度(IC_(50))值选择对缺氧环境适应能力较强的A549细胞,用同样方法进一步检测CoCl_(2)浓度为50、100、150和200μmol/L作用于细胞6、8、12、24和48 h的存活率。不同浓度CoCl_(2)作用同一时间,蛋白质印迹法检测细胞HIF-1α、c-Raf、MAPK和NF-κB的蛋白水平。Transwell小室检测CoCl_(2)对细胞迁移和侵袭能力的影响。结果当CoCl_(2)作用于A549细胞时,IC_(50)值为927.5μmol/L,作用于NCI-H1650细胞时,IC_(50)值为890.4μmol/L,成功建立缺氧NCI-H1650与A549细胞模型。当不同浓度CoCl_(2)(0~500μmol/L)作用于H1650细胞时,CoCl_(2)作用浓度对细胞存活率无影响,差异无统计学意义,F=2.942,P=0.058;作用于A549细胞时,细胞存活率分别为(1.05±0.04)%、(1.01±0.11)%、(0.81±0.05)%、(0.80±0.01)%、(0.77±0.02)%和(0.51±0.05)%,差异有统计学意义,F=30.930,P<0.001。其中,当浓度>100μmol/L时,各浓度组存活率均高于对照组,差异有统计学意义,均P<0.05。当CoCl_(2)作用时间为6~48 h,作用浓度一定时,A549细胞存活率无明显改变,差异无统计学意义;当CoCl_(2)作用时间不变,存活率随着浓度的增加而下降,并呈浓度依赖性,差异有统计学意义。与CoCl_(2)作用浓度0μmol/L[(38.33±7.64)个]相比,作用浓度分别为50、100、150、200μmol/L时A549细胞迁移数分别为(60.67±7.02)、(83.33±5.03)、(131.67±12.58)和(161.34±47.37)个,差异有统计学意义,F=127.671,P<0.001;其中,当CoCl_(2)浓度≥50μmol/L时迁移能力均�Objective To investigate the effects of hypoxia microenvironment on NF-kappa B pathway of Cmurraft MAPK in vitro,and to observe the effects of different concentrations of cobalt chloride(CoCl_(2))and different time on the proliferation,invasion and migration of human non-small cell lung cancer cells(NSCLC).Methods CoCl_(2)was used to establish an in vitro chemical hypoxia model for human NSCLC cell lines NCI-H1650 and A549 cells,with the action concentration of 0μmol/L as the control group and different concentrations as the experimental group.Different concentrations(100,200,300,400 and 500μmol/L)of CoCl_(2)acted on NCI-H1650 and A549 cells for 12 h,respectively.The proliferation level of NCI-H1650 and A549 cells was detected by CCK-8 method.The survival rate of A549 cells,which were more adaptable to the hypoxic environment,was selected according to the IC_(50) value;the survival rate of cells treated with CoCl_(2)at 50,100,150 and 200μmol/L for 6,8,12,24 and 48 h was detected by the same method.The protein levels of HIF-1α,c-Raf,MAPK and NF-κB were detected by western blotting at the same time of CoCl_(2)action at different concentrations,and the effect of CoCl_(2)on cell migration and invasion ability was examined in Transwell chambers.Results When CoCl_(2)was applied to A549 cells,the IC_(50) value was 927.5μmol/L,and when it was applied to NCI-H1650cells,the IC_(50) value was 890.4μmol/L.The hypoxic NCI-H1650and A549cell models were successfully established.When different concentrations of CoCl_(2)(0-500μmol/L)were applied to H1650 cells,there was no effect of CoCl_(2) on the cell survival rate,and the difference was not statistically significant,F=2.942,P=0.058.When CoCl_(2) was applied to A549 cells,the cell survival rates were(1.05±0.04)%,(1.01±0.11)%,(0.81±0.05)%,(0.80±0.01)%,(0.77±0.02)%and(0.51±0.05)%,respectively,and the differences were statistically significant,F=30.930,P<0.001.Among them,when the concentration was>100μmol/L,the survival rates of all concentration groups were higher

关 键 词：非小细胞肺癌 丝氨酸激酶 c-Raf/MAPK/NF-κB 增殖 侵袭 迁移 

分 类 号：R734.2[医药卫生—肿瘤]