023) and TTR (P = 0.014) (Supporting Information Table S4). We then stratified the 230 HCC patients by OPN expression and found that, in OPN+ patients, positive thrombin GDC-0941 in vivo expression (thrombin+/OPN+) was associated with a much shorter TTR compared with those of

thrombin− HCC (P < 0.0001; Fig. 3B). The 1-, 3-, and 5-year recurrence rates of thrombin+/OPN+ patients were 47.2, 86.1, and 88.9%, respectively, which were significantly higher than those of patients with thrombin−/OPN+ (20.4, 42.6, and 46.4%, respectively; P < 0.0001). The 1-, 3-, and 5-year OS rates of thrombin+/OPN+ patients (72.2, 27.8, and 22.2%, respectively) were significantly lower compared with those of thrombin−/OPN+ patients (85.2, 61.1, and 55.2%, respectively; P = 0.001). However, no significant difference in the survival and recurrence rates was observed between the thrombin− and thrombin+ patients within the OPN− group (TTR, P = 0.728; OS, P = 0.596; Fig. 3B). PLC cells were stably transfected with either wildtype OPN (PLC-OPN) or an empty vector control (PLC-CON); the OPN protein level of transfected cells was detected by western blot. PLC-OPN check details cells were confirmed to have an elevated level of OPN protein compared with PLC-CON cells (Supporting Information Fig. S3).

In vitro cell proliferation assays were performed to evaluate the difference between PLC-CON and PLC-OPN cells in response to thrombin treatment (2 U/mL). PLC-CON and PLC-OPN cells had similar growth kinetics in normal culture. When grown in the presence of thrombin, PLC-OPN cells demonstrated a selleck inhibitor significant increase in cell proliferation during the exponential growth phase (P < 0.05); however, PLC-CON cells demonstrated no significant proliferation changes when treated with thrombin (Fig. 4A). PLC-CON and PLC-OPN cells were evaluated for altered cell adhesion to various ECM molecules in response to thrombin (2 U/mL). Thrombin treatment significantly increased the adhesion of PLC-OPN cells, but not PLC-CON cells (Fig. 4B). As shown in Fig. 5A, both recombinant N-terminal fragment and full-length human OPN were able to significantly

increase the PLC-CON cell proliferation in the exponential growth phase (P < 0.05). The N-terminal fragment had a much stronger positive influence on proliferation than full-length OPN (P < 0.05). However, the recombinant C-terminal OPN fragment had no effect on PLC-CON cell proliferation (P > 0.05). To determine the effect of OPN on cell adhesion, purified OPN or its fragments were immobilized on microtiter plates and adhesion of PLC-CON cells to each recombinant protein was compared. As shown in Fig. 5B, more HCC cells adhered to N-terminal fragment of OPN compared with intact OPN (P < 0.05), whereas there was no significant adhesion to the C-terminal fragment or the negative control bovine serum albumin (BSA).