ANXA11 Regulates The Proliferation,Metastasis And Drug Resistances Of Human Hepatocellular Carcinoma Bel7402 Cells Via Cdc42 And Through Erk Signaling Pathway

Background: As a member of the annexin family,the dysregulation or mutation of annexin A11(annexin A11,ANXA11)is associated with the tumor metastasis,prognosis,invasion and drug resistance in ovarian cancer,breast cancer and colorectal cancer.Meanwhile,the previous study from our group showed that ANXA11 was closely associated with lymphatic metastatic potential of murine hepatocarcinoma cells.In this study,we aimed to gain more insight into the potential role of ANXA11 and the mechanism of action about regulating the malignant behaviors of Bel7402 cells.Objective: 1.pGPU6/GFP/Neo-sh RNA-Anxa11 vector was stably transfected into Bel7402 cells.The monoclonal Bel7402 cell lines with the stable down-regulation of ANXA11 were obtained;2.To investigate the effect of ANXA11 expression level on the proliferation,colony formation,migration,invasion,cell morphology,adhesion,cytoskeleton and drug resistance potential of Bel7402 cells;3.To explore the molecular mechanisms of ANXA11 regulating the malignant behaviors in Bel7402 cells.Methods: 1.The pGPU6/GFP/Neo-shANXA11 and pGPU6/GFP/Neo-sh Ctrl vector were transfected into Bel7402 cells.The monoclonal cell lines with ANXA11 knockdown were obtained by G418 screening selection.The expression level of ANXA11 were confirmed by q RT-PCR and Western Blot in monoclonal cells;2.MTT assay was determined to investigate the effect of ANXA11 knockdown on proliferation of Bel7402 cell;3.Colony formation assay was performed to investigate the role of ANXA11 silencing on the cell colony formatting capacity of Bel7402 cells;4.Microscope was applied to investigate the influence of ANXA11 knockdown on Bel7402 cell morphology;5.Transwell assay was measured to determine the influences of ANXA11 silencing on the migration and invasion abilities of Bel7402 cells;6.FITC-Phalloidin was performed to test the influence of ANXA11 downregulation on the F-actin cytoskeleton of Bel7402 cells;7.Lymph node cell adhesion and adhesion assay were performed to determine the effect of ANXA11 silencing on cell adhesion between cell and extracellular matrix,as well as cell and lymph nodes;8.MTT assay combined with Hoechst33258 staining were used to measure the influence of ANXA11 silencing on the drug-resistance and anti-apoptosis induced by;9.qRT-PCR,Western Blot and proteomics were performed to determine the molecular mechanisms of ANXA11 regulating Bel7402 malignant behaviors;10.si RNA-Cdc42 were transferred into monoclonal cell lines with ANXA11 stably down to determine whether ANXA11 regulates the metastasis of human hepatocellular carcinoma cell Bel7402 via Cdc42 molecular.Results: 1.The monoclonal cell lines with stable ANXA11 knockdown named as shANXA11-1 and shANXA11-2 were obtained by G418 screening.The expression levels of ANXA11 at m RNA level decreased by 73%(P=0.0016),63.8%(P=0.0029),at protein level decreased by 90%(P=0.0001),88%(P=0.0003);2.ANXA11 silencing suppressed the proliferation of Bel7402 cells compared with Bel7402-shCtrl cells;3.The colony formation ability decreased by 57.4% and 75%(P<0.0001)in monoclonal cell lines shANXA11-1,shANXA11-2 compared with Bel7402-shCtrl;4.The migration and invasion abilities respectively increased 2.3-,2.3-fold(P=0.0034,P=0.0015)and 3.4-fold(P=0.0012),3.3-fold(P=0.0066)in shANXA11-1 and shANXA11-2 compared with Bel7402-sh Ctrl;5.ANXA11 silencing made cell elongated by promoting F-actin cytoskeleton protein expression,increasing stress fibers and filopodia of Bel7402 cells,while,there was no difference between Bel7402 and Bel7402-sh Ctrl cells;6.ANXA11 knockdown promoted the adhesion ability of Bel7402 cell.The adhesion ability of shANXA11-1 and shANXA11-2 cells were significantly increased 0.4-(P<0.001)and 0.43-fold(P<0.001)compared to Bel7402-sh Ctrl cells,respectively,meanwhile,ANXA11 knockdown obviously promoted the lymphatic metastasis in Bel7402 cells.In shANXA11-1 and shANXA11-2 cells,the number of adhesive cells to LN were significantly increased 4.5-(P<0.001)and 6.4-fold(P<0.001)compared to Bel7402-sh Ctrl cells,respectively;7.ANXA11 knockdown enhanced drug resistances to cisplatin and 5-FU in Bel7402 cells;8.ANXA11 knockdown significantly inhibited Erk signaling pathway,conversely,MMP2,MMP9 and Cdc42 expression levels were increased obviously;9.Following ANXA11 silencing proteomic expression patterns were changed in Bel7402 cells.MRCK and Vinculin,as downstream effector of Cdc42,were obviously promoted;10.In shANXA11-2 cell line with ANXA11 stable down,cell adhesion,migration and invasion abilities were reversed,cytoskeletal proteins also enhanced by a reversal of depolymerization after transfecting si RNA-Cdc42.Conclusion: 1.The monoclonal Bel7402 cell lines with ANXA11 stable knockdown were obtained;2.ANXA11 knockdown inhibited the proliferation and colony formation of Bel7402 cells by inhibiting Erk signaling pathway;3.ANXA11 knockdown enhanced the potentials of invasion,migration and cell adhesion between cell-ECM,cell-LNM mainly through regulating Cdc42 to increase the formation of filopodia in Bel7402 cells;4.ANXA11 knockdown enhanced the drug resistances to cisplatin and 5-FU of Bel7402 cells.