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

To clarify whether 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.

HepG2 cells were stimulated and treated by gradient concentrations of lipopolysaccharide (LPS) and adriamycin, the supernatant 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.

The results of CLIA confirmed that the level of extracellular YB-1 in the supernatant 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).

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.