Effect Of ShRNA Mediated Down-regulation Of Annexin A2 On Biological Behaviour Of Human Lung Adencarcinoma Cells A549

Background and Objective:Lung cancer is a kind of malignant tumor which has higher level of incidence and worse therapeutic efficacy in the world, and has become the first cause of malignancy-related deaths in most countries. Malignant proliferation, invasion and metastasis are the essence for malignant phenotype of lung cancer, and the main factor impacting on survival rate and case fatality. Annexin A2 is a calcium-dependent phosphnolipid-binding protein that possesses many biology functions and is related to many diseases. A number of studies have discovered that Annexin A2 participates in tumor cells DNA synthesis, invasion, metastasis and relates to development of majority tumors. In previous work, we utilized serum and comparison proteome system to study the normal-metaplasia-dysplasia-carcinoma tissue of human bronchial epithelium, lung squamous cancer and normal tissue beside lung cancer, then discovered that the expression of Annexin A2 was obviously ascended in carcinoma tissue and possessed antigenicity. However the mechanism was unknown. In order to further investigate the relationship between Annexin A2 and lung cancer, our study is to apply RNA interference technique for researching the effect of silencing of Annexin A2 on biological behavior of human lung adencarcinoma cells A549, which provides experiment evidence for further illuminating molecule mechanism about malignant progression of lung cancer and searching potential target for prevention and treatment.Methods:(1) Human lung adencarcinoma cell line A549 as the research object, the shRNA plasmid targeting Annexin A2 was constructed in vitro and transiently transfected into A549 via lipofectamine 2000 mediation, at equal pace independence sequence control, keno-carrier control, blank control were set up, then transfection efficiency was observed by fluorescence microscope at 24 h posttransfection and shRNA plasmid interfering cells were harvested at 48 h posttransfection. (2) The mRNA expression difference of Annexin A2 was detected by semi-quantitative RT-PCR in every group cell. (3) The expression difference of Annexin A2 protein was examined by Western blotting and immuocytochemistry in every group cell. (4) The proliferation capability was determined by MTT assay. (5) Invasion capability of every group A549 cell in vitro was evaluated by using transwell chamber model. (6) The concentration difference of matrix metalloproteinase-2 and cathepsin B was measured by ELISA in the supernatant of every group cell.Results:(1) The shRNA plasmid targeting Annexin A2 was successfully transfected into human lung adencarcinoma cell line A549, and transfection efficiency was 60%. (2) At 48 h posttransfection, the expression of Annexin A2 mRNA and protein in shRNA plasmid transfection group was down-regulated significantly (p<0.05), (p<0.05), the proliferation capability of A549 cells descended extremely (p<0.05), the invasion capability of A549 cells in vitro decreased substantially (p<0.05), the concentration of matrix metalloproteinase-2 and cathepsin B in the supernatant reduced significantly (p<0.05), (p<0.05).Conclusions:(1) RNA interfering to Annexin A2 effectively degrades Annexin A2 mRNA and inhibits the expression of protein, and predominantly decreases proliferation and invasion capability of A549 cells in vitro. (2) Down-regulation of Annexin A2 gene expression can reduce the expression of downstream target genes including matrix metalloproteinase-2 and cathepsin B. (3) It implys that Annexin A2 might play an important role in the progression, invasion and metastasis of lung cancer, and regulating the expression of matrix metalloproteinase-2 and cathepsin B could be one of pathways for the effect of Annexin A2 on biological behavior of human lung adencarcinoma cells A549.