Y-box结合蛋白1通过细胞外调节蛋白激酶信号通路介导肝癌细胞对索拉非尼耐药  

Y-box-binding protein 1 mediates sorafenib resistance via the extracellular signal regulated-protein kinase pathway in hepatoma cells

作　　者：刘婷 谢肖立[1] 陈圣雄[1] 王一军 姜慧卿[1] Liu Ting;Xie Xiaoli;Chen Shengxiong;Wang Yijun;Jiang Huiqing(Department of Gastroenterology,the Second Hospital of Hebei Medical University,Hebei Key Laboratory of Gastroenterology,Hebei Institute of Gastroenterology,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学第二医院消化内科、河北省消化病实验室、河北省消化病研究所,石家庄050000

出　　处：《中华肝脏病杂志》2023年第4期401-407,共7页Chinese Journal of Hepatology

基　　金：国家自然科学基金面上项目(81770601);河北省研究生创新资助项目(CXZZBS2019121)。

摘　　要：目的探讨Y-box结合蛋白1(YB-1)在肝癌细胞对索拉非尼耐药中的作用及其可能的机制。方法分别构建过表达及敲低YB-1的慢病毒载体,单独或联合索拉非尼刺激人肝癌细胞株HepG2、Huh7细胞。过表达部分实验共计分为4组:过表达对照组(Lv-NC)、YB-1过表达组(Lv-YB-1)、过表达对照联合索拉非尼孵育组(Lv-NC+sorafenib)、YB-1过表达联合索拉非尼孵育组(Lv-YB-1+sorafenib)。敲低部分实验也分为4组:敲低对照组(Lv-shNC)、YB-1敲低组(Lv-shYB-1)、敲低对照联合索拉非尼孵育组(Lv-shNC+sorafenib)、YB-1敲低联合索拉非尼孵育组(Lv-shYB-1+sorafenib)。末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记染色检测细胞凋亡的发生情况,蛋白质印迹法检测细胞外调节蛋白激酶(ERK)信号通路的关键蛋白磷酸化(p)-ERK、ERK的蛋白表达水平,并通过ImageJ软件进行定量分析。进行裸鼠皮下成瘤实验,并予索拉非尼治疗,在体内验证YB-1对索拉非尼疗效的影响。2组数据之间的比较采用独立样本t检验;3组及以上数据间的比较应用单因素方差分析。结果索拉非尼可以促进肝癌细胞凋亡的发生,而过表达YB-1抑制细胞凋亡,同时还可以抵抗索拉非尼对凋亡的促进作用;相反,敲低YB-1可促进细胞凋亡,还可以增强索拉非尼对细胞凋亡的诱导作用。此外,索拉非尼孵育可下调p-ERK的水平(HepG2:Lv-NC 0.685±0.143,Lv-NC+sorafenib 0.315±0.168,P<0.05;Huh7:Lv-NC 0.576±0.078,Lv-NC+sorafenib 0.150±0.131,P<0.01),过表达YB-1可抵抗索拉非尼所诱导的p-ERK的降低(HepG2:Lv-NC+sorafenib 0.315±0.168,Lv-YB-1+sorafenib 0.688±0.042,P<0.05;Huh7:Lv-NC+sorafenib 0.150±0.131,Lv-YB-1+sorafenib 0.553±0.041,P<0.05);而敲低YB-1可进一步增强索拉非尼引起的p-ERK下调(HepG2:Lv-shNC+sorafenib 0.911±0.252,Lv-shYB-1+sorafenib 0.500±0.201,P<0.05;Huh7:Lv-shNC+sorafenib 0.577±0.082,Lv-shYB-1+sorafenib 0.350±0.143,P<0.05),并在裸鼠体内得到进一Objective To investigate the effect and possible mechanism of Y-box-binding protein 1(YB-1)on sorafenib resistance in hepatoma cells.Methods Lentiviral vectors with YB-1 overexpression and knockdown were constructed,respectively,to stimulate human hepatoma cell lines(HepG2 and Huh7)alone or in combination with sorafenib.The overexpression part of the experiment was divided into four groups:overexpression control group(Lv-NC),YB-1 overexpression group(Lv-YB-1),overexpression control combined with sorafenib resistance group(Lv-NC+sorafenib),YB-1 overexpression combined with sorafenib resistance group(Lv-YB-1+sorafenib).The knockdown part of the experiment was also divided into four groups:knockdown control group(Lv-shNC),YB-1 knockdown group(Lv-shYB-1),knockdown control combined with sorafenib resistance group(Lv-shNC+sorafenib),YB-1 knockdown combined with sorafenib resistance group(Lv-shYB-1+sorafenib).The occurrence of cell apoptosis was detected by TUNEL.The protein expression levels of phosphorylated(p)-ERK and ERK,key proteins in the extracellular regulatory protein kinase(ERK)signaling pathway,were detected by Western blot and quantified by ImageJ software.Subcutaneous tumorigenesis experiments were performed in nude mice.The effect of YB-1 on the efficacy of sorafenib was verified in vivo.The comparison between the two sets of data was carried out by an independent sample t-test.One-way ANOVA was used for comparisons between the three groups of data above.Results Sorafenib had accelerated the occurrence of apoptosis in hepatoma cells,while YB-1 overexpression had inhibited cell apoptosis,and at the same time also inhibited the apoptosis-accelerating impact of sorafenib.On the contrary,YB-1 knockdown accelerated cell apoptosis and amplified the induction effect of sorafenib on apoptosis.Furthermore,sorafenib resistance had down-regulated p-ERK levels(HepG2:Lv-NC 0.685±0.143,Lv-NC+sorafenib 0.315±0.168,P<0.05;Huh7:Lv-NC 0.576±0.078,Lv-NC+sorafenib 0.150±0.131,P<0.01),whereas YB-1 overexpression had inhibi

关 键 词：肝细胞癌 细胞外调节蛋白激酶 索拉非尼 耐药性 Y-box结合蛋白1 

分 类 号：R735.7[医药卫生—肿瘤]