Figure 3.

HCAL Regulates HCC Cell Proliferation, Apoptosis, Migration, and Invasion In Vitro and In Vivo

(A) Relative HCAL expression in SMMC-7721 and MHCC-97h cells transduced with lentiviruses expressing control shRNA (Con), HCAL shRNA1 (sh1), or shRNA2 (sh2) was determined by performing qPCR. (B) Proliferation of control and HCAL-knockdown cells was assessed by performing the CCK-8 assay. HCAL knockdown suppressed the proliferation of SMMC-7721 and MHCC-97h cells. (C) Colony-formation assays were performed using control and HCAL-knockdown cells. Left, crystal violet staining; right, number of colonies from three independent experiments. (D) HCAL-knockdown cells were treated with 5-fluorouracil (100 mg/mL) for 48 hr, stained with annexin V and PI, and analyzed by flow cytometry. Annexin V-positive cells were designated as apoptotic cells. Percentage of apoptotic cells is shown. (E) Cleaved PARP1 and caspase-3 levels after HCAL silencing were determined by performing western blot analysis. (F) Left, representative images of the migration and invasion of SMMC-7721 and MHCC-97h cells expressing control and HCAL shRNAs. Right, statistical results obtained from three independent experiments. (G) Effects of HCAL knockdown on HCC growth in vivo. Upper panel, representative images of tumors formed in nude mice subcutaneously injected with HCAL-knockdown SMMC-7721 cells; lower panel, tumor growth curves measured after injecting SMMC7721 cells expressing control and HCAL shRNAs. (H) Representative images of pulmonary and intrahepatic metastases in nude mice subcutaneously injected with control and HCAL-knockdown SMMC-7721 cells obtained by performing HE staining. Data are expressed as mean ± SD; *p < 0.05.