| Background: Lymphatic metastasis is the early stage of metastases of malignantepithelial tumors. The mechanism of tumor lymphatic metastasis is unclear. Hca-F andHca-P, a pair of mouse hepatocarcinoma cell lines with different potential lymph nodemetastasis rates (Hca-F>75%, Hca-P<25%) were proven to be ideal models for studyingthe process of lymphatic metastasis. Quantitative proteomics from our group indicatedthat Anxa3was up-regulated to2.3-fold in Hca-F of that in Hca-P, which was validatedby gene chip result. Anxa3is a member of the annexin family that are Ca2+-dependantphospholipid-binding proteins. Accumulated recent study indicated that the abnormalexpressions of Anxa3were associated with ovarian cancer, lung adenocarcinoma,prostatic carcinoma, hepatocarcinoma, renal carcinoma, colorectal cancer andpancreatic cancer, however, the detailed action mechanisms of Anxa3in tumors areunclear. Our previous work indicated that Anxa3might functionalized in lymphaticmetastasis of mouse hepatocarcinoma. In current work, we explored thedown-regulation of Anxa3by RNAi on the malignant behaviors of Hca-F.Objective:1. pGPU6/GFP/Neo-shRNA-Anxa3expression plasmids wereconstructed and transfected into Hca-F cells. The shRNA-Anxa3stably transfectedHca-F cells were then obtained.2. To study the cell proliferation, cell adhesion, cellmigration and cell invasion ability, cell cycle and apoptosis status and cell morphologychanges of Hca-F cells following the down-regulation of Anxa3.Methods:1.Three shRNAs targeting different sites of the Anxa3gene weredesigned and inserted into pGPU6/GFP/Neo vectors, named aspGPU6/GFP/Neo-shRNA-Anxa3-175, pGPU6/GFP/Neo-shRNA-Anxa3-380andpGPU6/GFP/Neo-shRNA-Anxa3-874respectively. Negative control of unrelated sequence was also designed. All the expression plasmids were validated by BamH I andPst I and confirmed by DNA sequence analysis. Then the threepGPU6/GFP/Neo-shRNA-Anxa3expression vector as well as the unrelated sequenceexpression vector were transfected into Hca-F cell using SofastTMtransfection reagent.And the transfection efficiencies were determined by fluorescence microscopy after24h-transfection. The corresponding stably transfected cells were then obtained byscreening in G418(400g/ml) for3weeks. The expression levels of Anxa3at mRNAand protein levels were measured by real time quantitative polymerase chainreaction(qRT-PCR) and Western Blot analysis for Hca-F, pGPU6/GFP/Neo-shRNA-Anxa3transfected Hca-F and negative control pGPU6/GFP/Neotransfected Hca-F cells. The strongest suppression efficient shRNA and thecorresponding transfected Hca-F cell line was then selected for further experiment.2.The Anxa3level was found more reduced in shRNA-Anxa3-380transfected Hca-F thatwas chosen and named as shRNA-Anxa3-Hca-F for further experiments. Cells weredivided into three groups, Hca-F, negative control Hca-F, shRNA-Anxa3-Hca-F cells.Cell proliferations were determined by cell counting kit-8(CCK-8) assay. Cell adhesionability was measured using the CytoSelectTM48-well extracellular matrix (ECM) array(Cell Biolabs, USA), with kits that contained collagen I, collagen IV, fibrinogen,fibronectin and laminin. The adhesion abilities of Hca-F and shRNA-Anxa3-Hca-F tomouse lymph nodes were also determined. The capabilities of migration and invasion ofthe different cell lines were measured by Transwell chamber assay. Flow cytometryanalysis was performed to analyze the cell cycle and apoptosis.Results:1. The ttarget sequences inserted into pGPU6/GFP/Neo expressionplasmids were successfully confirmed correct by DNA sequence analysis. The level ofmRNA expression of Anxa3in shRNA-Anxa3-380-Hca-F was decreased by72%compared to Hca-F. Anxa3protein level in shRNA-Anxa3-380-Hca-F was reduced50%compared to Hca-F.2. Results from CCK-8assay indicated that the cell proliferationwas reduced by25%following Anxa3knock-down in96h. Morphology observationindicated that following Anxa3suppression, Hca-F cell grew dispersedly and the celledge became uneven. The downregulation of Anxa3did not cause notable alteration ofthe cell cycle. However, the cell number of early apoptosis decreased significantly.While, we found this phenomenon was caused due to the alteration of cell membraneattributed by the exposure and translocation of phosphatidylserine(PS) and decreasedstability of cell membrane. The migration and invasion capabilities of shRNA-Anxa3-Hca-F cells decreased by48%and67%due to the downregulation ofAnxa3. Cell adhesion assay: Adhesive capability of shRNA-Anxa3-Hca-F cells tolymph node and Collagenâ… decreased by72.2%and36.5%respectively compared toHca-F cells by the downregulation of Anxa3.Conclusion:1. Three pGPU6/GFP/Neo-shRNA-Anxa3expression vectors aswell as negative control vector were constructed successfully and transfected intoHca-F cells stably. pGPU6/GFP/Neo-shRNA-Anxa3-380expression vector was confirmed to be more efficiency than others, Hca-F cells transfected pGPU6/GFP/Neo-shRNA-Anxa3-380expression vector were chosen to be object of study forfurther experiments.2. The down-regulation of Anxa3inhibited proliferation of Hca-F cells, but did not affect the cell cycle status; The down-regulation of Anxa3destoryed the stablity of membrane, the membrane was everted and eventuallycaused the cell number of the early apoptosis increasing evidently. With the down-regulation of Anxa3, the capability of migration and invasion were inhibited, the adhesive capability to lymphy node and Collagenâ… decreased. |
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