胞外Y-盒结合蛋白1通过Notch3受体促进肝癌细胞HepG2的增殖与转移

Extracellular Y-box binding protein-1 promotes proliferation and metastasis of HepG2 cells through Notch3 receptor

目的 阐明应激状态下肝癌细胞HepG2是否主动分泌Y-盒结合蛋白1（YB-1）,并初步探讨胞外YB-1的病理意义与作用机制. 方法 浓度梯度的脂多糖（LPS）、阿霉素刺激处理HepG2,离心收集培养上清液,采用已建立的化学发光免疫分析法（CLIA）实时定量检测上清液中YB-1水平;免疫共沉淀技术检测胞外YB-1是否特异结合Notch3受体,Western blot分析Notch-跨腹受体表达;浓度梯度的重组YB-1与HepG2共培养,四甲基偶氮唑盐法与细胞迁移实验检测HepG2细胞的增殖与侵袭转移.多组间数据比较采用单因素方差分析. 结果 CLIA检测结果显示培养液上清液中胞外YB-1水平显著高于对照组（F=10.54,P＜0.001）,分泌表达量在刺激培养（LPS：8 ng/ml和阿霉素：10 ng/ml）4 h后达高峰;免疫共沉淀与Westem blot结果证实胞外YB-1特异结合并上调表达Notch3;四甲基偶氮唑盐法与细胞迁移实验表明胞外YB-1显著促进HepG2细胞的增殖与侵袭转移（F=9.405,P＜0.001）.结论 化疗药物等应激状态下肿瘤细胞HepG2可经非经典途径主动分泌YB-1,胞外YB-1特异结合并上调表达Notch3受体,进而促进HepG2细胞的增殖与侵袭转移.为阐明肝细胞肝癌发病机制及进一步探讨胞外YB-1与恶性肿瘤的生物学关系奠定了基础.

Objective To clarifywhether HepG2 cells actively secrete Y-box binding protein-1 （YB-1） under stress conditions,and to investigate the pathological significance and mechanism of action of extracellular YB-1.Methods HepG2 cells were stimulated and treated by gradient concentrations of lipopolysaccharide （LPS） and adtiamycin,the supematant of the culture solution was collected by centrifugation,and the established chemiluminescence immunoassay （CLIA） was used for real-time quantitative determination of YB-1 level in the supernatant.The co-immunoprecipitation assay was used to detect whether extracellular YB-1 specifically bound to Notch3 receptor,and Western blot was used to measure the expression of Notch-NICD.The gradient concentrations of recombinant YB-1 were co-cultured with HepG2 cells,and MTT and migration assays were used to analyze the proliferation and invasion/metastasis of HepG2 cells.One-way analysis of variance was used for comparison of data between multiple groups.Results The results of CLIA confirmed that the level of extracellular YB-1 in the supematant was significantly higher than that in the control group （F =10.54,P 〈 0.001）,and the secretory expression of YB-1 reached its peak after 4 hours of stimulation （LPS：8 ng/ml;adriamycin：10 ng/ml）.The results of co-immunoprecipitation assay and Western blot showed that extracellular YB-1 specifically bound to Notch3 receptor and upregulated the expression of the Notch3 receptor.MTT and migration assays showed that extracellular YB-1 significantly promoted the proliferation and invasion/metastasis of HepG2 cells （F =9.405,P 〈 0.001）.Conclusion Under the stress conditions induced by chemotherapeutics,HepG2 cells can actively secrete YB-1 via non-classical pathways.Extracellular YB-1 can specifically bind to Notch3 receptor and further up-regulate its expression,and then promote the proliferation and invasion/metastasis of HepG2 cells.This study lays a foundation for further clarifying the pathogenesis of hepatocellular carcinoma and investigating the biological relationship between extracellular YB-1 and malignant tumors.