The Selective Expression In Non-small Cell Lung Cancer Of Annexin A2 And Its Induction In Lung Cancer Cell Invasion And Metastasis

IntroductionAnnexins are associated closely with genesis and development in different kinds of tumors.Annexin A2 is a member of the Annexins family,it is a calcium-dependent, phospholipid-binding protein found on many cell types.It consists of a short hydrophobic tail,which dictates its function,and a core domain,which is involved in phospholipid binding.Annexin A2 has been implicated in a number of biochemical processes,including signal transduction,cellular motility,DNA synthesis,cell proliferation and apoptesis. Annexin A2 has been shown to be dysregulated in many neoplasfic diseases.The expression levels of which is associated closely with the genesis,infiltration,metastasis, clinical stage and prognosis of tumor.So far,there were few reports on invasion and metastasis of Annexin A2 in non-small cell lung cancer.The purpose of this study is to find out the expression of Annexin A2 in non-small cell lung cancer and its effects on invasion and metastasis in the lung cancer cells in vitro.Materials and Methods1 Materials and reagents120 cases of primary pulmonary squamous cell carcinoma,adenocarcinoma in paraffin-embedded specimens which were obtained No.1 Hospital of China Medical University between 2000 to 2005.Normal bronchial epithelial cells HBE,human lung giant cell carcinoma cell linc BE1(high invasion subtype),LH7(low invasion subtypes);Lung adenocarcinoma cell line A549,A2,LTEP-a-2(LTE) and SPC-A-1 (SPC).Mouse anti-human Annexin A2 monoclonal antibody(sc-28385) purchased from Santa Cruz company,SP high-sensitivity kit(Kit-9701) purchased from Fuzhou step development of new biotechnology companies,rabbit anti-human polyclonal anti-β-actin antibody(sc-1616) purchased from Zhongshan Biotechnology Co.,Ltd., horseradish peroxidase labeled goat anti-mouse antibody(ZB-2305),goat anti-rabbit secondary antibody(ZB-2301) purchased in Beijing Jinqiao Biotechnology in Sequoia Limited company.Annexin A2 siRNA(sc-29199) purchased from Santa Cruz, transfection reagent(lipofectamine 2000 reagent) purchased from Invitrogen Corporation.2 Methods(1) Cell culture(2) Immunohistochemistry(S-P)Neutral formalin-fixed specimens by the conventional paraffin-embedded,4μm thick serial sections,the use of streptomycin avidin-peroxidase method(S-P),carried out immunohistochemical staining,and dipping with DAB.With PBS instead of primary antibody as a negative control.(3) Immunohistochemistry(S-P) of lung cancer cell linesAfter cells fixed by adding liquid,the use of streptomycin avidin-peroxidase method(SP),carried out immunohistochemical staining.And dipping with DAB, brown reaction product shows the antigen targeting with PBS instead of primary antibody as a negative control.(4) Western blotExtraction of cell protein,using brilliant blue Kamaz protein quantitative method. Then it followed by the whole procedure,including electrophoresis,transfer,blocking, incubation with primary antibody and enzyme conjugated compound,ECL luminescence,the results of gel electrophoresis images was analyzed by automatic analyzer imaging.Repeat the experiment 3 times,taking an arithmetic mean value for statistical analysis.(5) SiRNA transfectionTransfections were performed using Lipofectamine 2000 in serum-free medium. Lipofectamine 2000 and siRNA were diluted separately in serum-free medium at a ratio of 1:25 and incubated at room temperature for 5 minutes.Equal volumes of siRNA and Lipofectamine 2000 solutions were mixed and incubated at room temperature for 15 minutes.Monolayers were washed and placed in serum-free medium media.Transfection solutions were diluted 1:5 into cultures for a final siRNA concentration of 80 nmol/L.After incubation overnight,the monolayers were placed into complete media.(6) Wound healing experimentLTE cells in 6-well culture plates were transfected at 65%confluency with siRNA. Three days after siRNA transfection,when cells reached 100%confluency,a single linear wound was created through the monolayer with a sterile pipette tip.Sites at which wounds were to be measured were marked on the undersurface of the wells to ensure that measurements were taken at the same place.Wounds were imaged at 0,12 and 24 hours,Ten measurements along the wound length were averaged to determine the wound width.(7) Statistical analysisThe statistical software SPSS 13.0 was applied for data analysis.T-test were used while P＜0.05 was significative in statistic.Results1 In the adjacent noncancerous tissue,there is high expression of Annexin A2 in cilia of normal bronchial epithelial cells;The expression of annexin A2 was correlated closely with clinic stage(P=0.021),lymph node metastasis(P=0.030) and differentiation deagree(P=0.000),but had no relationship with gender(P=0.441),age (P=0.703) and historical type(P=0.844).2 The level of Annexin A2 expression in lung cancer cell lines is not consistent, giant cell carcinoma cell lines is relatively high,and the expression of Annexin A2 in adenocarcinoma cell lines is relatively low;3 The expression of Annexin A2 in HBE is in the nucleus and the cytoplasm, mainly in the nucleus;The expression of Annexin A2 in LTE is in cytoplasm and nucleus,mainly in cytoplasm;The expression of Annexin A2 in BE1 is in membrane and cytoplasm;4 LTE cells were growing slowly after the transient transfection of Annexin A2 SiRNA;Their migration and proliferation ability was significantly reduced.ConclusionThe high expression of Annexin A2 is related to malignant degree and invasion of lung cancer;Annexin A2 expresses selectively and induces invasion and metastasis in non-small lung cancer cell lines.