抑制成纤维细胞生长因子受体2-卷曲螺旋结构域蛋白6融合基因表达在人胆管癌异种移植小鼠的作用  

Effect of inhibiting the expression of fibroblast growth factor receptor-2-coiled coil domain containing 6 fusion gene on human cholangiocarcinoma xenografts in nude mice

作　　者：王绍清 王宇[2] 李鸿梅 张宁 吴甜 Wang Shaoqing;Wang Yu;Li Hongmei;Zhang Ning;Wu Tian(College of Pathology,Qiqihar Medical University,Qiqihar 161003,China;Department of Hepatobiliary Surgery,Southern Hospital of Southern Medical University,Guangzhou 516006,China)

机构地区：[1]齐齐哈尔医学院病理学院,齐齐哈尔161006 [2]南方医科大学南方医院肝胆外科,广州516006

出　　处：《中华实验外科杂志》2023年第7期1319-1321,共3页Chinese Journal of Experimental Surgery

基　　金：齐齐哈尔医学科学院项目(QMSI2017B-05);齐齐哈尔市科技局社会发展攻关项目(SFGG201626)。

摘　　要：目的探究抑制成纤维细胞生长因子受体2-卷曲螺旋结构域蛋白6(FGFR2-CCDC6)表达在人胆管癌异种移植小鼠的抗肿瘤作用。方法选择购自美国模式培养物集存库(ATCC)的人正常肝内胆管上皮细胞HIBEC和稳定转染FGFR2-CCDC6表达质粒的人肝内胆管癌细胞系Hucct1, 分组为沉默FGFR2-CCDC6阴性对照(si-NC)组和沉默FGFR2-CCDC6(si-FGFR2-CCDC6)组。利用实时定量聚合酶链反应(RT-PCR)检测融合基因表达;蛋白质印迹法(Western blot)检测FGFR2、成纤维细胞生长因子2(FGF2)和磷酸化细胞外调节蛋白激酶(p-ERK1/2)蛋白表达。噻唑蓝(MTT)检测抑制FGFR2对细胞增殖的抑制作用。小鼠荷瘤模型探究抑制FGFR2表达对移植瘤体积的抑制作用。应用SPSS 21.0软件进行分析。结果 Hucct1中的FGFR2-CCDC6表达显著高于HIBEC(1.00±0.05比1.92±0.09, t=15.480, P<0.05);FGFR2、FGF2和p-ERK1/2的蛋白表达均明显高于HIBEC(FGFR2:0.54±0.03比1.29±0.05, t=20.360, FGF2:1.32±0.06比1.87±0.09, t=9.207, p-ERK1:0.84±0.05比1.54±0.07, t=14.920, p-ERK2:1.42±0.06比2.11±0.11, t=9.391, 均P<0.05)。si-FGFR2-CCDC6组的FGFR2-CCDC6的mRNA表达水平显著低于si-NC组(1.00±0.06比0.39±0.03, t=19.540, P<0.05);FGFR2、FGF2和p-ERK1/2的蛋白表达均显著低于si-NC组(FGFR2:1.32±0.05比0.77±0.04, t=13.080, FGF2:1.93±0.08比1.23±0.07, t=11.12, p-ERK1:1.48±0.07比0.73±0.06, t=15.260, p-ERK2:2.03±0.12比1.44±0.07, t=7.488, 均P<0.05);转染后24 h和48 h细胞增殖率明显低于si-NC组(24 h:0.74±0.06比0.48±0.04, t=6.245, 48 h:0.97±0.07比0.65±0.06, t=6.012, 均P<0.05)。小鼠荷瘤模型发现, si-FGFR2-CCDC6转染荷瘤组移植瘤体积明显小于si-NC参照组(598.00±50.00比476.00±39.00, F=12.530, P<0.05)。结论抑制FGFR2-CCDC6融合基因表达可抑制FGF2/FGFR2/p-ERK1/2通路的激活, 抑制细胞增殖和肿瘤生长, 进而发挥抑癌作用。Objective To explore the antitumor effect of inhibiting fibroblast growth factor(FGF)receptor 2(FGFR2)-coiled-coil domain containing 6(FGFR2-CCDC6)expression in human cholangiocarci-noma xenografts in nude mice.Methods Human normal intrahepatic bile duct epithelial cells(HIBECs)and human intrahepatic cholangiocarcinoma cell line Hucctl with stable transfection of FGFR2-CCDC6 from American Type Culture Collection(ATCC)were selected to construct silencing FGFR2-CCDC6 negative con-trol(si-NC)group and silencing FGFR2-CCDC6(si-FCFR2-CCDC6)group.Real-time polymerase chain re-action(RT-PCR)was used to detect the expression of the fusion gene.The protein expression of FCFR2,FGF2 and phosphorylated extracellular regulated protein kinases 1/2(p-ERK1/2)was detected by Western blotting.The inhibitory effect of inhibiting FGFR2 expression on cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay,and corresponding inhibitory effect on the transplanted tumor volume was further explored through the mouse tumor-bearing model.Results FCFR2-CCDC6 expression in Hucctl was significantly higher than that in HIBECs(1.00±0.05 vs.1.92±0.09,t=15.480,P<0.05);and the expression levels of FGFR2,FGF2 and p-ERK1/2 proteins were significantly increased(FCFR2:0.54±0.03 vs.1.29±0.05,t=20.360;FCF2:1.32±0.06 vs.1.87±0.09,t=9.207;p-ERK1:0.84±0.05 vs.1.54±0.07,t=14.920;p-ERK2:1.42±0.06 vs.2.11±0.11,t=9.391,all P<0.05).The expression of FGFR2-CCDC6 in si-FGFR2-CCDC6 group was significantly lower than that in si-NC group(1.00±0.06 vs.0.39±0.03,t=19.540,P<0.05).The expression levels of FGFR2,FCF2 and p-ERK1/2 proteins decreased significantly(FCFR2:1.32±0.05 vs.0.77±0.04,t=13.080;FGF2:1.93±0.08 vs.1.23±0.07,t=11.120;p-ERK1:1.48±0.07 vs.0.73±0.06,t=15.260;p-ERK2:2.03±0.12 vs.1.44±0.07,t=7.488,all P<0.05).The cell proliferation rate decreased significantly at 24 h and 48 h after transfection(24 h:0.74±0.06 vs.0.48±0.04,t=6.245;48 h:0.97±0.07 vs.0.65±0.06,t=6.012,all P<0.05).The tumor bearing-mice model showed tha

关 键 词：胆管癌 成纤维细胞生长因子受体2-卷曲螺旋结构域蛋白6 融合基因 异种移植 

分 类 号：R735.8[医药卫生—肿瘤]