| Background:As an important feature of malignant tumors, metastasis is the leading death causeof most cancer patients. Tumor cells disseminate in body through llymphatic metastasisand blood metastasis. Studies have indicated that the initial metastases of mostmalignant tumors were through lymphatic instead of blood vessel. Consequently, thelymphatic metastasis can be used as the indicator of the early metastasis of tumors.Whether lymphatic metastasis happens or not will critically determine the optimaloperative treatment strategy for patients. However, rare study has been made on themolecular mechanism for tumor lymphatic metastasis.Hca-F and Hca-P are a pair of mouse hepatocarcinoma cell lines with same geneticbackground and different lymphatic metastatic potentials. The lymph node metastaticrates for Hca-F and Hca-P are about~75%and~25%, respectively, which make them asthe ideal models for studying the lymphatic metastasis of tumors. As a member ofAnnexin family, annexin A5(Anxa5) has been raveled de-regulated in colorectal cancer,prostate cancer, liver cancer, squamous cell carcinoma and cervical cancer byaccumulated recent studies. However, few studies have been made on the associationAnxa5with tumor lymphatic metastasis.Previous proteomic results from our group indicated that the protein level of Anxa5in Hca-F was3.16-fold of that in Hca-P, which was consistent with its differentialexpression levels at gene level, which also implicates that Anxa5is a potential targetmolecule for promoting the lymphatic metastasis of hepatocarcinoma. In current work,the association of Anxa5with tumor cell lymphatic metastatic behaviors were exploredby down-regulating the expression level of Anxa5in Hca-F cell by RNA interferencetechnique. Objective:1. To construct the PGPU6/GFP/Neo-shRNA-Anxa5expressionplasmids, to stably transfect the constructed plasmids into Hca-F cells and screen theHca-F cell lines with knock-down of Anxa5;2. To reveal the changes of cellproliferation, cell migration, cell invasion ability and cell cycle of Hca-F following theprotein down-regulation of Anxa5.Methods:1. Three shRNA targeting the sequence sites352,792and958weredesigned according to Anxa5gene sequence provided in GenBank. One unrelatedsequence was also designed as the negative control (NC). The three correspondingpGPU6/GFP/Neo-shRNA-Anxa5and pGPU6/GFP/Neo-NC expression vectors wereconstructed and confirmed by sequence determination. Then, the constructed expressionplasmids were stably transfected into Hca-F cells using SofastTMtransfection reagent.And the transfections were observed using fluorescence microscope and the transfectionrates were calculated in24h following the transfection. The successfully transfectedHca-F cells were cultured and screened in media containing G418with theconcentration of400g/ml until the monoclonal transfected Hca-F cells were obtained.The expression levels of Anxa5in screened monoclonal shRNA-Anxa5andunrelated-sequence-transfected Hca-F cells as well as in Hca-F cells were determinedby Western blotting. According to the RNAi efficacy, the monoclonalshRNA-Anxa5-transfected Hca-F cells with varied expression levels of Anxa5werechosen for further experiments.2. The cell proliferation, cell invasion and cell migrationcapacities, and cell cycles of monoclonal shRNA-Anxa5-transfected Hca-F cells weredetermined by cell counting kit-8(CCK-8) assay, Transwell chamber assay and flowcytometry (FCM) and compared with those of unrelated-sequence-transfected Hca-Fcells and Hca-F cells, respectively.Results:1. Anxa5targeted RNA interference recombinant plasmids wereconstructed successfully as the sequence analysis was exactly matched the designedones. Eighteen monoclonal cells were screen from three sets of shRNA-Anxa5recombinant plasmids. Among them, the monoclonal Hca-F cells originated frompGPU6/GFP/Neo-shRNA-Anxa5-729group were named as1-1,1-2,1-3,1-4,1-5and1-6; the monoclonal cells from pGPU6/GFP/Neo-shRNA-Anxa5-958group werenamed as2-1,2-2,2-3,2-4,2-5and2-6; and the monoclonal cells frompGPU6/GFP/Neo-shRNA-Anxa5-352group were named as3-1,3-2,3-3,3-4,3-5and3-6, respectively. Western blotting analysis indicated that the3-3and3-5monoclonalHca-F cells showed the optimal interfering efficacies with the Anxa5suppression rate of ~100%, the1-1and2-2monoclonal cells showed~80.2%and~63.8%decreases forAnxa5protein expression levels. The1-1,2-2and3-3cell lines were chosen for furtherexperiments.2. CCK-8assay indicated that Anxa5protein down-regulated to certainextent could apparently inhibit the proliferation of Hca-F cells. Characterization fromTranswell chamber assay showed that the migration and invasion capacities of Hca-Fcould be inhibited by protein down-regulation of Anxa5closely related to thedown-regulation degree of Anxa5. Characterization from flow cytometry analysisindicated that cell cycle phase could be mediated by the down-expression of Anxa5gene.Conclusion:1. Three pGPU6/GFP/Neo-shRNA-Anxa5expression plasmids targetingthree targeting sequence sites of Anxa5were successfully constructed. Eighteenmonoclonal Hca-F cell lines were obtained with varied siRNA interference efficiencies.2. The cell proliferation, migration and invasion capacities could be inhibited by theprotein reduction of Anxa5closely related to its down-regulation extent. Thedown-expression of Anxa5shortens the G0/G1phase and extends G2/M phase of Hca-Fcells by arresting cells at G2/M phase. |
