The Role Of N-cadherin In Human Hepatocellular Carcinoma Invasion And Metastasis And Its Mechanism

Aim: To investigate the expression of N-cad in HCC tissues and the relationships between the expression of N-cad and its clinicopathologic features as well as surgical outcomes after liver resection.Methods: The HCC samples and their adjacent liver tissues were collected from 64 HCC patients who underwent hepatic resection and the normal liver tissues were acquired during hepatectomy for hepatic cavernous hemangioma in 5 patients. The expression of N-cad was measured by immunohistochemistry and western blot. The relationships between the expression of N-cad and its Clinicopathologic features as well as postoperative survival were studied retrospectivly.Results: N-cad expression was found strong in the membranes of hepatocytes in the normal liver tissues and adjacent liver tissues, but lost or reduced in HCC tissues. Among the 64 HCC, 34 (53%) HCC were classified as the reduced expression type (RdE). In poorly differentiated tumors, 25/33 (76%) of tumors indicated decreased N-cad expression; in the moderately differentiated tumors, 6 of 19 (32%) of these tumors exhibited decreased N-cad staining; in the well-differentiated HCC, 9 of 12 (75%) showed strongly and uniformly N-cad staining at the cell-cell boundaries in all tumors. The frequency of RdE in poorly differentiated tumors was significantly higher than that in well and moderately differentiated tumors (P < 0.01). Tumor thrombus and invasion in portal vein or hepatic vein branches was significantly correlated with reduced N-cad expression (92%, 11/12). But, no association was found between reduced N-cad expression and other clinical parameters such as age, sex, HBV infection, presence of cirrhosis and tumor size. Furthermore, reduced N-cad expression was significantly correlated with shorter disease-free survivals(P<0.05), although no significant difference was found in overall survival rates between the patients with preserved N-cad expression and the patients with loss or reduced N-cad expression(P=0.108) Conclusions:The expression of N-cad was reduced in HCC tissues compared with its adjacent liver tissues, and reduced N-cad expression was correlated with poor differentiation, vascular invasion and poor postoperative disease-free survival of HCC. Aim: To study the effect of N-cad on cell proliferation, cell adhesion, cell invasion and metastasis with HCCLM3 cells in vitroMethods: To establish the stable N-cad-knockdown HCCLM3 with RNA interference;to invesigate the effects of N-cad on cell proliferation by directly cell counting, on cell adhesion by cell aggregation assay, on cell invasion and metastasis by Transwell motility and invasion assayResults: The stable cell clones with N-cad knockdown were established, and western blot indicated that N-cad expression was almost suppressed in the clone Ncad1, Ncad2, Ncad7 and Ncad9 expressing shRNA against endogenous N-cad, but not in the control clones eGFP1 and eGFP2 and HCCLM3 cells. There was no effect of N-cad knockdown on HCCLM3 cell proliferation. The cell aggregation assay indicated that large cell aggregates were formed in the control clone eGFP1, but not in the Ncad2 and Ncad7 clones. The aggregation indexes for the Ncad2 and Ncad7 were increased by 24% and 27%, compared to 11% for the control clone eGFP1 (P < 0.01). There was no difference in cell aggregation index between Ncad2 and Ncad7 . In cell motility and invasion assays, Ncad2 and Ncad7 clone cells showed a strong increase in migration comparing to control clone eGFP1 (P < 0.01), and also increased ability to invade through Matrigel-coated filters (P < 0.01). There was no difference between cell motility and invasion for N-cad2 and N-cad7.Conclusions: N-cad knockdown in HCCLM3 cells decreased cell-cell adhesion and increased cell motility and invasion. Aim: To explore the molecular mechanism underlied the role of N-cad knockdown in promoting tumor cell invasion and metastasisMethods: The expression ofβ-catenin in N-cad-knockdown cell clones was evaluated by immunofluorescence and western blot; the target gene of Wnt/β-catenin, cyclin D1 was detected by western blot; in addition, the expressions of N-cad andβ-catenin in HCC tissues were measured by immunohistochemistry and the relationship between them was studied.Results: By immunofluorescence study, no nuclearβ-catenin staining was found in N-cad-knockwon cells. However,β-catenin staining in membranes was largely decreased in the N-cad-knockdwon cells, comparing with that in N-cad-expressing cells. Western blot showed that the totalβ-catenin expression was reduced in N-cad-knockdwon cells,and the expression of cyclin D1 had no change. In HCC tissues, the reduced expression ofβ-catenin on cell membranes was correlated with reduced N-cad expression.Conclusions: Wnt/β-catenin pathway was not activated in N-cad-knockdwon cells, and the detailed mechanism for increased cell mitility and invasion in N-cad-knockdwon cells needs to be further studied. Howerver, decreased cell adhesion may be parlty responsible for it. Aim: To establish a HCC metastasis model so as to provide a suitable model for the following studyMethods: 5×106 of the HCCLM3 cells were injected subcutaneously into nude mice and the tumor volum was evalated periodically. When the subcutaneous tumor had reached approximately 1 cm in diameter, the tumor was removed and cut into pieces of approximately 1×1×1mm3,which were implanted into the livers of another nude mice. The mice were killed and studied by histopathology under microscope following HE staining.Results: When the cells were injected subcutaneously into nude mice for 10 days, the tumor nodules usually became visible or palpable. After 2 weeks, the tumor can reached approximately 1 cm in diameter. After orthotopic implantation of tumor tissues, the mice were killed on day 35, 42 and 60. We observed large tumors in the sites of implantation in livers, but on spontaneous lung or intrahepatic metastases on day 35. Huge tumors in the sites of implantation in liver as well as spontaneous lung metastases were detected on day 42 and 60.Conclusions: After orthotopic implantation of tumor tissue into nude mouse liver for 42 days, spontaneous lung metastasis occurred.