高尔基体磷蛋白3通过整合素信号通路调控肝内胆管癌干细胞特性  

Golgi phosphoprotein 3 promoting the cancer stemness of intrahepatic cholangiocarcinoma by regulating integrins pathway

作　　者：陈高辉 朱志文 阳亮芳 郑杨 何惠娟 黄海丽 包仕廷 杨春燕 戴维 Chen Gaohui;Zhu Zhiwen;Yang Liangfang;Zhang Yang;He Huijuan;Huang Haili;Bao Shiting;Yang Chunyang;Dai Wei(Department of Hepatobiliary Surgery,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524001,China;Clinical Medicine Research Center,Affiliated Hospital of Guangdong Medical University,Zhanjiang 524001,China;Surgical Laboratory,Clinical Skills Training Center,Guangdong Medical University,Zhanjiang 524001,China)

机构地区：[1]广东医科大学附属医院肝胆外科,湛江524001 [2]广东医科大学附属医院临床医学研究中心,湛江524001 [3]广东医科大学临床技能培训中心外科实验室,湛江524001

出　　处：《中华实验外科杂志》2023年第10期1966-1970,共5页Chinese Journal of Experimental Surgery

基　　金：广东省自然科学基金(2022A1515012568);湛江市科技计划项目(2022A01182)。

摘　　要：目的探究高尔基体磷蛋白3(GOLPH3)对肝内胆管癌干细胞特性的影响及其机制。方法利用TCGA数据库(35例胆管癌组织和9例正常组织)分析GOLPH3在胆管癌中的表达。免疫组织化学分析25例胆管癌根治手术患者癌组织和癌旁组织中GOLPH3的表达模式。将GOLPH3敲低质粒和对照质粒转入肝内胆管癌细胞系(HuCCT1),荧光显微镜和蛋白质印迹法(Western blot)验证转染效率。集落形成实验检测细胞增殖能力。肿瘤球形成实验和Western blot检测干细胞样特性。样本数据采用非配对t检验。结果TCGA数据库中胆管癌组织的GOLPH3表达高于正常组织(P<0.001)。敲低GOLPH3抑制了HuCCT1细胞的增殖能力和干细胞特性,对照组的细胞克隆数目高于敲低组[(175.72±16.04)个比(67.33±5.69)个,t=11.024,P<0.01];对照组的肿瘤球平均直径高于敲低组[(244.43±38.82)μm比(131.32±19.86)μm,t=4.593,P<0.01];单层细胞中对照组的SOX2蛋白表达水平高于敲低组(0.75±0.07比0.39±0.08,t=4.304,P<0.05);单层细胞中对照组的CD44蛋白表达水平与敲低组比较,差异无统计学意义(0.31±0.11比0.23±0.09,t=0.916,P>0.05);球形细胞中对照组的SOX2蛋白表达水平高于敲低组(0.85±0.04比0.54±0.02,t=12.357,P<0.01);球形细胞中对照组的CD44蛋白表达水平高于敲低组(0.81±0.09比0.57±0.06,t=7.841,P<0.05)。京都基因与基因组百科全书(KEGG)富集分析和基因本体论(GO)注释提示GOLPH3的致癌作用与整合素通路激活有关。GOLPH3敲低后整合素(ITG)/局部黏着斑激酶(FAK)通路活性下降,单层细胞中对照组的ITGA2蛋白表达水平高于敲低组(0.70±0.07比0.42±0.09,t=30.404,P<0.01);单层细胞中对照组的ITGB1蛋白表达水平高于敲低组(0.68±0.10比0.42±0.04,t=6.735,P<0.05);单层细胞中对照组的p-FAK蛋白表达水平高于敲低组(0.61±0.04比0.39±0.04,t=22.084,P<0.01);球形细胞中对照组的ITGA2蛋白表达水平高于敲低组(0.82±0.08比0.56±0.05,t=13.485,P<0.Objective To explore the effect of golgi phosphoprotein 3(GOLPH3)on the characteristics of intrahepatic cholangiocarcinoma stem cells and its mechanism.Methods The expression of GOLPH3 in cholangiocarcinoma was analyzed by TCGA database(35 cases of bile duct cancer tissue and 9 cases of normal tissue).Immunohistochemistry was used to analyze the expression pattern of GOLPH3 in tumor tissue and adjacent tissues of 25 patients with cholangiocarcinoma undergoing radical surgery.The GOLPH3 knockdown plasmid and control plasmid were transferred into the intrahepatic cholangiocarcinoma cell line(HuCCT1),and the transfection efficiency was verified by fluorescence microscopy and Western blotting.Colony formation test was used to detect the cell proliferation ability.Tumor globular formation test and Western blotting were used to detect the stem cell-like characteristics.Unpaired t-test was used for sample data.Results GOLPH3 expression was higher in cholangiocarcinoma tissues than in normal tissues in the TCGA database,P<0.001.Knockdown of GOLPH3 inhibited the proliferative ability and stem cell properties of HuCCT1 cells,and the number of cell clones was higher in the control group than in the knockdown group:175.72±16.04 vs.67.33±5.69,t=11.024,P<0.01;The average diameter of tumor spheres in the control group was higher than that in the knockdown group:(244.43±38.82)μm vs.(131.32±19.86)μm,t=4.593,P<0.01;the SOX2 protein expression level of the control group in monolayer cells was higher than that of the knockdown group:0.75±0.07 vs.0.39±0.08,t=4.304,P<0.05;CD44 protein expression level in the control group in monolayer cells was higher than that in the knockdown group:0.31±0.11 vs.0.23±0.09,t=0.916,P>0.05;SOX2 protein expression level in the control group in spherical cells was higher than that in the knockdown group:0.85±0.04 vs.0.54±0.02,t=12.357,P<0.01;CD44 protein expression level in control group was higher than that in the knockdown group in spherical cells:0.81±0.09 vs.0.57±0.06,t=7.841,P<0.05.KEGG

关 键 词：肝内胆管癌 高尔基体磷蛋白3 肿瘤干细胞 整合素 

分 类 号：R735.8[医药卫生—肿瘤]