Anxa5 Mediating The Malignant Behaviors Of Mouse Hepatocarcinoma Cell Line Hca-F Through ERK1/2 Pathway

Background: Maligant tumor with early lymph node metastasis leads to poor prognosis and high mortality rate of patients, its mechanisms is always a difficult problem of tumorigenesis. Annexin A5(Anxa5) is one member of annexins family, which belongs to Ca2+-regulated phospholipid-binding proteins. Accumulated studies indicated that Anxa5 deregulation closely correlates with tumor progression, metastasis and drug resistance. Our group previous found that Anxa5 overexpressed in the mouse HCC high lymphatic metastatic Hca-F cell line by proteomic and gene array methods, and obtained two monoclonal cells Hca-F-sh RNA1 and Hca-F-shRNA2 with stable knockdown of Anxa5 by siRNA, furthermore, Anxa5 downregulation inhibited proliferation ability of Hca-F cells. The above result implied a potential promoting role of Anxa5 in the lymphatic metastasis of HCC.Objective: 1. To study the effects of Anxa5 stably knockdown on the migration, invasion and adhesion ability of Hca-F cells. 2. To investigate the molecular mechanisms of Anxa5 regulating the proliferation, migration, invasion and adhesion of Hca-F cells, providing theory basis and therapeutic targrt for the diagnosis and treatment of HCC.Methods: 1. Quantitative real-time reversed transcription-PCR(qRT-PCR) and Western blot were performed to detect the expression level of Anxa5 in Hca-F-shRNA1 and Hca-F-shRNA2 cells; 2. Transwell chamber and in situ cell lymphatic adhesion assay were used to measure the effects of Anxa5 downregulation on migration, invasion and adhesion abilities of Hca-F cells; 3. q RT-PCR and Western blot were used to detect the effects of Anxa5 downregulation on proliferation and metastasis related molecules c-jun, JNK, ERK, p38 MAPK, AKT, MEK and E-cadherin expression; 4. Western blot was performed to detect the effects of Anxa5 downregulation on c-jun and E-cadherin expression after treatment with ERK inhibitor PD98059.Results: 1. Compared with Hca-F-shControl, the Anxa5 mRNA and protein expression level of Hca-F-shRNA1 and Hca-F-shRNA2 cells reduced 72.3% and 97.1%, 43.9% and 99.7%(p<0.05), respectively; 2. Compared with Hca-F-shControl, the migration and invasion capacities of Hca-F-sh RNA2 cell decreased 50.71% and 49.84%(p<0.05), respectively, while Hca-F-shRNA1 cell had no effect(p>0.05); 3. Compared with Hca-F-sh Control, the lymph node adhesion capacity of Hca-F-shRNA2 cell decreased 61.71%(p<0.05); 4. The expression of p-ERK2 downregulated 92.7% in Hca-F-shRNA2 cell(p<0.01), while p-ERK1, JNK, p38-MAPK, AKT and MEK unchanged; 5. The expression of p-c-jun(Ser73) decreased 68.3% in Hca-F-shRNA2 cell(p<0.05); 6. The mRNA and protein expression of E-cadherin increased 2.53-fold(p<0.05) and 8.08-fold(p<0.001) in Hca-F-shRNA2 cell, respectively; 7. PD98059 significantly inhibited p-ERK and p-c-jun(Ser73) expression of Hca-F-sh Control and Hca-F-shRNA2 cells, meanwhile, promoted E-cadherin expression of Hca-F-shControl cell, but had no effect on E-cadherin expression of Hca-F-shRNA2 cell.Conclusion: 1. Anxa5 down-regulation to a certain degree could inhibit the migration and invasion abilities of Hca-F cells; 2. Anxa5 down-regulation could reduce lymph node adhesion capacity of Hca-F cells in vitro which indicate that Anxa5 downregulaton could inhibit the lymph node metastasis ability of Hca-F cells in vivo; 3. Anxa5 downregulation inhibited Hca-F cell proliferation by reduceing ERK and p-c-jun(Ser73) expression instead of JNK; 4. Anxa5 downregulation promoted E-cadherin expression via inhibiting ERK to reduce migration and invasion abilities of Hca-F cells; 5. Anxa5 mediating the malignant behaviors of mouse hepatocarcinoma cell line Hca-F through ERK1/2 pathway.