产甲胎蛋白型胃癌中甲胎蛋白表达的调控机制及其与肿瘤细胞恶性生物学行为的关系  

Regulatory mechanism of alpha fetoprotein expression in alpha-fetoprotein-producing gastric cancer and its relationship with malignant phenotype

作　　者：梅彦侠 李明[1] 文继航 李军[1] 孔祥兴[1] Mei Yanxia;Li Ming;Wen Jihang;Li Jun;Kong Xiangxing(Department of Colorectal Surgery,the Second Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310009,China)

机构地区：[1]浙江大学医学院附属第二医院大肠外科,浙江杭州310009

出　　处：《实用肿瘤杂志》2023年第6期528-536,共9页Journal of Practical Oncology

基　　金：国家自然科学基金面上项目(82172851);国家自然科学基金青年项目(82103684,11932017)。

摘　　要：目的探究产甲胎蛋白型胃癌(alpha-fetoprotein-producing gastric cancer,AFPGC)细胞中甲胎蛋白(alpha fetoprotein,AFP)表达的调控机制及其与肿瘤细胞恶性生物学行为的关系。方法使用人AFPGC细胞系FU97,采用亚克隆分选技术,建立18个亚克隆细胞株(M1~M18)。Western blot及荧光实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测AFP表达,挑选出AFP表达最高和最低的2个亚克隆细胞株。通过划痕实验、上皮间质转化(epithelialmesenchymal transition,EMT)分子标志物检测、药物毒性实验及NSG小鼠皮下成瘤实验比较2个亚克隆细胞株的恶性生物学行为。应用RNA-seq检测FU97-AFP^(High)和FU97-AFP^(Low)细胞的差异表达基因,同时京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析关键信号通路改变,并使用Yes关联蛋白1(Yes-associated protein 1,YAP1)小分子抑制剂verteporfin干预实验进行验证。结果Western blot显示,FU97细胞不同亚克隆株AFP的表达水平差异大,亚克隆株M8和M12分别有最低和最高的AFP蛋白表达量。RT-qPCR显示,AFP高表达细胞株M12(FU97-AFP^(High))和AFP低表达细胞株M8(FU97-AFP^(Low))的AFP表达量分别为(38.70±3.37)和(3.33±0.04),差异具有统计学意义(P<0.01)。FU97-AFP^(High)细胞的迁移能力大于FU97-AFP^(Low)细胞(P<0.01),且高表达N-cadherin,低表达E-cadherin。细胞毒性实验表明,FU97-AFP^(High)细胞对5-氟尿嘧啶、多西他赛、顺铂和伊立替康的半抑制浓度(half maximal inhibitory concentration,IC50)分别为87.57、43.49、18.11和2.43μmol/L,均大于FU97-AFP^(Low)细胞,后者分别为77.19、38.14、16.76和0.94μmol/L。小鼠皮下成瘤实验显示,接种第40天时FU97-AFP^(High)细胞注射点形成皮下瘤,而FU97-AFP^(Low)注射点未见成瘤迹象。RNA-seq及富集分析显示,FU97-AFP^(High)细胞的磷酯酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)-Akt、Hippo-YAP、Wnt和转化生长�Objective To explore the regulatory mechanism of alpha fetoprotein(AFP)expression in alpha-fetoprotein-producing gastric cancer(AFPGC)cells and the relationship between AFP expression and tumor malignant phenotype.Methods Using human AFPGC cell line FU97,eighteen monoclones(M1-M18)were established by subclonal sorting technology.Western blot and fluorescence real-time quantitative polymerase chain reaction(RT-qPCR)were used to detect AFP expression.Monoclones with the highest and lowest AFP expression were selected and notated as FU97-AFP^(High)and FU97-AFP^(Low),respectively.Wound-healing assay,epithelial-mesenchymal tran-sition(EMT)biomarker detection,cytotoxicity experiment and subcutaneous NSG mouse model assay were performed to compare the malignant phenotypes of two monoclones.RNA-seq was used to detect the genes with differential expressions in FU97-AFP^(High)and FU97-AFP^(Low)cells,and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment was performed to reveal the pivotal changes of signaling pathways,the result of which was validated by using verteporfin,a small molecular inhibitor of Yes-associated protein 1(YAP1).Results Western blot showed significant differences in AFP expression among M1-M18 monoclones,and monoclones M8(FU97-AFP^(Low))and M12(FU97-AFP^(High))had the minimum and maximum AFP expressions.RT-qPCR showed that the AFP expressions of FU97-AFP^(Low)and FU97-AFP^(High)monoclones were(38.70±3.37)and(3.33±0.04),respectively(P<0.01).The migration of FU97-AFP^(High)cells was greater than FU97-AFP^(Low)cells(P<0.01),with higher expression of N-cadherin and lower expression of E-cadherin protein.Cytotoxicity assay showed that the half maximal inhibitory concentrations(IC50)of FU97-AFP^(High)cells for 5-fluorouracil,docetaxel,cisplatin,and irinotecan were 87.57,43.49,18.11 and 2.43μmol/L,higher than those of FU97-AFP^(Low)cells which were 77.19,38.14,16.76 and 0.94μmol/L,respectively.Subcutaneous NSG mouse model assay showed tumor formation at the injection point of FU97-AFP^(High)cells on

关 键 词：产甲胎蛋白型胃癌 甲胎蛋白 亚克隆分选 恶性表型 Hippo-YAP 

分 类 号：R735.2[医药卫生—肿瘤]