Cck 8 method

The cell viability was measured using MTT assay (Fisher, China) at 24 h, 48 h, 72 h, and 96 h of the culture period. After that, the cells were incubated for another 4 h at 37°C following by 150 mL dimethyl sulfoxide (Sigma) treatment for 10 min. The cells from each group were collected and evaluated for the proliferation assay using CCK‐8 method (Fisher). The absorbance was evaluated at 450 nm using the plate reader purchased from Thermo Fisher Scientific Co. Meanwhile, the cell apoptosis was measured using flow cytometry after Annexin V FITC/PI double staining.

The corresponding TME prognostic gene siRNAs (50 nmol/L) (OriGene) and the corresponding negative control transfection were obtained by Lipofectamine-2000 (Sigma-Aldrich, USA).
The siRNA-transfected cells were harvested and the proliferation ability was measured using the CCK-8 method (Fisher, China). At the same time, the apoptosis ability of the cells was examined by the method of flow cytometry after Annexin V FITC/PI double staining. For transwell migration assays, 2.5×10⁴ cells were seeded in the appropriate serum-free medium into the pre-coated upper chamber. The 500-μl complete medium was used as chemoattractant in the lower chambers. The incubation time was set at 48 h, after which cells without migration or invasion were removed. The final number of migrated or invaded cells was examined using Image-Pro Plus version 6.0 software. All experiments were repeated three times independently.

The HCC cell line HCCLM3 was purchased from ATCC Co. For siRNA transfection, specific LRP1B, CDCA8 and centromere protein A (CENPA) siRNAs (50 nmol/l) (OriGene) and a negative control were utilized. Lipofectamine 2000 (Sigma–Aldrich, U.S.A.) was used for transfection. The cells from each group were collected and evaluated for the proliferation assay using CCK-8 method (Fisher, China). The absorbance was evaluated at 450 nm using the plate reader purchased from Thermo Fisher Scientific Co. Meanwhile, the cell apoptosis was measured using flow cytometry after Annexin V-FITC/PI double staining. All experiments were repeated three times independently.