Annexin A2 Promotes Migration And Invasion Of Human Hepatocellular Carcinoma Cells In Vitro Via Regulating Shedding Of Cd147-harboring Microvesicles From Tumor Cells

【Background】Hepatocellular carcinoma (HCC) is one of the most common malignancies, and metastasis is the may reason for HCC-related death. Migration and invasion are critical procedures in tumor metastasis. The dysregulation of oncogene and anti-oncogene and changes of tumor local microenviroment are involved in the process.Annexin A2 (ANXA2) is a Ca2+-dependent phospholipid-binding protein with multiple biological functions. Increasing genomic and proteomic studies have begun to accumulate evidence about the differential expression of ANXA2 in malignant neoplasms of diverse orgin. And the possible involvement and association of ANXA2 with malignant transformation, tumor invasion and metastasis are being investigated. Three independent studies from different countries found that expression of ANXA2 is upregulated in HCC, and plays important role in the transformation process from normal liver cells to tumor cells[2-4]. But the role of ANXA2 in migration and invasion of HCC remains obscure. In the present study, we knocked down the expression of ANXA2 in HCC cells by RNAi to explore its role in HCC migration and invasion.CD147 is a tumor-related molecule belonging to the immunoglobulin superfamily. And one of the most important functions of CD147 is to stimulate the production of matrix metalloproteinase (MMPs). Recent studies have provided evidence that microvesicles shedding from tumor cells carry full-length CD147 and play a role in tumor–stromal interactions through upregulation of MMPs production[5, 9]. Binding with membrane is another role of ANXA2, and ANXA2 has been reported to be implicated in many membrane-related functions, including membrane and microvesicle trafficking. Published literature speculated that there may exist certain interactions between ANXA2 and CD147, but the hypothesis has not been testified to date[8]. Base on the backroud listed above, we hypothesize that ANXA2 may be involved in the shedding of CD147-harboring microvesicles from HCC cells, and thus regulates tumor migration and invasion.【Aims】To explore the role of ANXA2 in migration and invasion of human hepatocellular carcinoma cells in vitro and the possible mechanism underlying the phenomenon.【Methods】1) HCC cells (SMMC-7721 and FHCC-98) were transfected with siRNA using LipofectAMINETM 2000 reagent according to the manufacturer's instruction; 2) RT-PCR and Western Blot were employed to evaluate the interference efficiency of ANXA2 specific siRNA (si-ANXA2); 3) HCC cells and fibroblasts (human embryo pulmonary fibroblast-1, HPF-1) were co-cultured in the upper compartment of the Millicell chamber, and in vitro migration/invasion assays were used to explore the effects of ANXA2 on migration and invasion of HCC; 4) Gelatin zymography was used to study the role of ANXA2 in regulating the production of MMPs by HCC cells and HPF-1; 5) Expression and distribution of ANXA2 and CD147 were illustrated by immunocytochemistry; 6) Total cellular membrane proteins (TMP) were extracted from HCC cells, and the interactions of ANXA2 with CD147 in TMP were detected by ProFound? Mammalian Co-Immunoprecipitation Kit (anti-JEV mAb was used as a control); 7) Microvesicles shedding from HCC cells were isolated by ultracentrifugation and analysed under an electron microscopy; 8) The isolated microvesicles were analyzed by Western Blot; 9) SMMC-7721 cells were treated with snc-RNA, si-ANXA2 or si-CD147, and then microvesicles were isolated from SMMC-7721; 10) Western Blot was engaged to explore the role of ANXA2 in the releasing of CD147-harboring microvesicles; 11) Gelatin zymography was used to study the effects of CD147-harboring MV of diverse origin on the production of MMPs from HPF-1.【Results】1) ANXA2 specific siRNA down-regulated ANXA2 expression effectively in HCC cells; 2) Downregulation the expression of ANXA2 in HCC cells inhibited migration and invasion potential of HCC cells co-cultured with fibroblasts in vitro; 3) MMP-2 production was significantly decreased in HPF-1 cultured in supernant collected from si-ANXA2-transfected HCC cells; 4) ANXA2 and CD147 co-localized in SMMC-7721 and FHCC-98 cells; 5) TMP were successfully extracted from SMMC-7721 and FHCC-98 cells, and ANXA2 and CD147 were found co-immunoprecipitated with each other in TMP extracted from both cell lines; 6) MV were isolated and both ANXA2 and CD147 were detected in MV by Western Blot; 7) Expression of CD147 in MV was downregulated when HCC cells were transfected with si-ANXA or si-CD147; 8) CD147-harboring MV of diverse origin enhanced the production by HPF-1, and the effects were weakened when HCC cells were transfected with si-ANXA or si-CD147.【Conclusions】1) ANXA2 promotes the migration and invasion of HCC cells co-cultured with fibroblasts, and ANXA2 acts as an important molecule in the hepatocarcinogenesis and HCC development; 2) ANXA2 promotes the production of MMP-2 by fibroblasts via regulating the release of CD147-harboring microvesicles from HCC cells, and thus accomplishes tumor-stroma crosstalk, which facilitates the migration and invasion of HCC cells.