Figure 4. Effects of NKCC1 overexpression/knockdown and inhibitor treatment on the growth and extrahepatic metastasis of HCC cells in vivo.

(A) Sizes of tumors formed from subcutaneously injected NKCC1-overexpressing MHCC97L cells and control MHCC97L cells transfected with empty vector. (B) Sizes of tumors formed from subcutaneously injected NKCC1 knockdown MHCC97H cells and control MHCC97L cells transfected with shRNA-NC. Tumor growth was monitored every week by measuring the tumor diameter (mean±SD). (A-a) and (B-a) Photographs of all xenograft tumors in mice. (A-b) and (B-b) Statistical results of tumor volume. (C) The effect of bumetanide on MHCC97H cell xenotransplantation in vivo. Water (negative control), 350 mg/kg of sorafenib (positive control), or bumetanide (1 mg/kg, 2 mg/kg, or 4 mg/kg) were administered by oral gavage. Tumor size (C-a) and tumor weight (C-b) in MHCC97H subcutaneously inoculated nude mice are shown (bars indicate average value). (D) NKCC1-overexpressing MHCC97L and control cells or (E) NKCC1-knockdown MHCC97H and control cells were injected into the tail veins of nude mice. Lung metastasis was checked after 16 weeks. (D-a) The number of the lung metastatic nodules in NKCC1 overexpression group was compared with that in the control group. (D-b) Histological images of lung metastatic nodules formed from MHCC97L cells. (E-a) The number of lung metastatic nodules in the NKCC1 knockdown group was compared with that in the control group. (E-b) Histological images of lung metastatic nodules formed from MHCC97H cells. * p<0.05 or ** p<0.01 indicates a significant difference (two-tailed Student's t-test). hNKCC1, overexpression of NKCC1; NC, negative control; shNKCC1, knockdown of NKCC1 with shRNA.