The Investigation Of Anxa11Mediating The Malignant Behaviors Of Mouse Hepatocarcinoma Cell Line Hca-P And Corresponding Molecular Mechanism

Background: Lymphatic metastasis and drug-resistance of hepatocarcinomacancer (HCC) cell are important factors that affect on the prognosis and survival ofpatients. The studies on the lymphatic metastatic mechanism and drug-resistance canprovide basis for the clinical diagnosis and treatment of liver cancer. Annexin A11(Anxa11) is a member of annexins superfamily of Ca2+-regulated phospholipid-bindingproteins. Accumulated studies showed that the dysregulation of Anxa11was closelyassociated with tumor cell malignant transformation, chemoresistance and metastasis,however, the specific molecular mechanism remains unclear. Mouse hepatocarcinomacell Hca-P with low metastatic potential (~25%) and Hca-F with high metastaticpotential (~75%) are a pair of synogenetic hepatocellular carcinoma ascites cell lines,those two cell lines sharing same genetic background are ideal experimental subjects forthe study of lymph node metastasis (LNM) of liver cancer. Previous Western blot resultsfrom our group indicated that the expression level of Anxa11in Hca-P was about2-foldhigher than that of Anxa11in Hca-F cells, which indicated that Anxa11might be apotential protein to inhibit the lymphatic metastasis of HCC.Objective:1. To construct pGPU6/GFP/Neo-shRNA-Anxa11expression vectorsand abtain monoclonal Hca-P cell (P-1) with Anxa11down-regulated stably;2. Toinvestigate the influence of Anxa11downregulation on apoptosis, migration, invasion,adhesion and chemoresistance properties of Hca-P cell in vitro;3. To determine theeffects of Anxa11downregulation on the lymph node metastatic abilities of Hca-P in vivo; and4. To investigate Anxa11downregulation on the expressions of transcriptionalfactor c-Jun and its phosphorylated form before or after the treatments of drugs in Hca-Pcells.Methods:1. The Anxa11siRNAs were designed according to the mRNA sequenceof Anxa11. A non-targeting sequence was also designed as a negative control.2. Thereconstructed pGPU6/GFP/Neo-shRNA-1,2and negative control expression vectorswere validated by restriction enzyme digestion and DNA sequence analysis.3. Thereconstructed expression vectors stably transfected into Hca-P cells using Sofasttransfection agent. The monoclonal Hca-P cells with stable knockdown of Anxa11werescreened by G418selection and limited dilution. The expression levels of Anxa11inmonoclonal Hca-P cell lines were confirmed by Western blot and qRT-PCR;4.Transwell chamber assay and in situ cell lymphatic adhesion assay were performed toassay the effect of Anxa11downregulation on the migration, invasion and adhesionabilities of Hca-P cells in vitro;5. Mouse foot-pad subcutaneous injection method wasutilized to investigate the effect of Anxa11downregulation on the lymph nodemetastasis of Hca-P cell in vivo;6. Western blot was used to analyze the influence ofAnxa11knockdown on the expression level of corresponding apoptotic factors of Hca-Pcells;7. Cell counting kit-8(CCK-8) assay and Hoechst33258staining methods weremeasured to test the influence of Anxa11downregulation on the chemoresistance andantiapoptotics induced by5-FU for Hca-P cells;8. Western blot was performed to detectthe influence of Anxa11downregulation on the expressions of transcriptional factorc-Jun and its phosphorylated form following the treatments of various concentrations of5-FU in Hca-P cells.Results:1. The pGPU6/GFP/Neo-shRNA-1,2and negative control expressionvectors were constructed successfully;2. Monoclonal Hca-P cell line (P-1) with Anxa11stable knockdown was obtained. qRT-PCR and Western blot showed that the expressionlevels of Anxa11in P-1cells significantly downregulated by~82.49%and80.53%compared with P-nc cells, respectively.3. The migration, invasion and adhesion abilitiesof P-1cell line increased~1.55,~1.20and~1.13(~1.35)-folds compared to P-nc cells, respectively;4. The in vivo results showed that lymph node metastasis capacity of P-1cell line were higher than that of P-nc cell line;5. The downregulation of Anxa11hadno effects on apoptosis but increased the expression of Bax and Bcl-2of Hca-P cells;6.P-1cell line presented distinctly chemoresistance for5-FU, but not cisplatin;7.Compared with P-nc cells, the pSer73-c-Jun was increased in P-1cells without thetreatment with5-FU and the expression levels of c-Jun and pSer73-c-Jun in P-1cellswere up-regulated after treatment with5-FU in a dose-dependent manner.Conclusion：1. The pGPU6/GFP/Neo-shRNA-1,2and negative control expressionvector were constructed successfully;2. Monoclonal Hca-P cell line with stableknockdown of Anxa11were obtained;3Anxa11downregulation increases the in vitromigration, invasion and lymphatic adhesion capacities of Hca-P cell;4. Anxa11downregulation promotes Hca-P cell lymphatic metastasis capacities in vivo;5. Anxa11downregulation increases the expression of Bax and Bcl-2of Hca-P cells.6. Anxa11downregulation enhances5-FU chemoresistance of Hca-P cells;7. Anxa11downregulation potentially regulates the lymphatic metastasis and chemoresistance ofHca-P cells by affecting the expressions of c-Jun and phosphorylated c-Jun (Ser73).