The Corelation Of The Expression Of Annexin A7 And The Potential Of Lymphatic Metastasis Of Mouse Hepatocarcinoma Cell

Background: Metastasis is the fundamental cause of the high mortality of the patients with malignant tumors. Lymphatic metastasis is the most important factor which affects the prognosis of carcinoma. So, it is very important to make clear about the molecular mechanisms of lymphatic metastasis and find new approaches to treating it .Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines, presenting a specific potential of lymphatic metastasis when inoculated subcutaneously in 615 mice. Hca-F with a high metastasis potential (>70%) and Hca-P with a low one (<30%) are rare and available cell model as a reference each other in the reseach on the molecular mechanisms of lymphatic metastasis.We have screened out the lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using cDNA microarray and gene chip assays respectively, and obtained the lymphatic metastasis-associated proteins by using quantitative proteomics technique. Based on the results of these studies we found that both in mRNA level and in protein level Annexin A7 expression was higher in Hca-F than that in Hca-P cells. Therefore, we focus our attention upon the corelation of the Annexin A7 expression and lymph node metastasis of mouse hepatocarcinoma.Objiective:①Up-regulating the Annexin A7 expression in Hca-P cell line by constructing the pCDNA3.1- Annexin A7 expressing vectors to transfect the Hca-P cell stably.②Down regulating the Annexin A7 expression in Hca-F cells by using pSilencer 3.1-shRNA expressing vectors to transfect the Hca-F cell stably.③. The corelation of the Annexin A7 expression and lymphatic metastasis of mouse hepatocarcinoma was evaluated based on the change of cell cycle, apoptosis rate, potential of migration and invasion of Hca-P and Hca-F, due to the change of the level of Annexin A7 expression.Methods:①The Annexin A7 gene was amplified by PCR. BamH I and EcoR I enzyme were used to digest the Annexin A7 gene and PcDNA3.1 plasmid. The pcDNA3.1- Annexin A7 expressing vectors was constructed to transfect the Hca-P stably. In the end, the effection of transfecting was tested by genome DNA checkup and western-blotting.②Three shRNAs were designed and inserted into the pSilencer vector to silence Annexin A7 gene. The three pSilencer 3.1-shRNA expressing vectors were transfected into Hca-F cells respectively, and the most effective pSilencer 3.1-shRNA vector was selected based on the results of RT-PCR and Western blotting. In the end, the Hca-F cells transfected stably with the most effective pSilencer 3.1-shRNA expressing vector were obtained and the effection of RNAi was tested by Western blotting.③the cell viability, cell cycle, apoptosis rate, potential of migration and invasion of The Hca-P and The Hca-F were evaluated by MTT, flow cytometry,and transwell assays.Results:①The DNA sequencing result showed that the pcDNA3.1- Annexin A7 was constructed successfully; Genome DNA checkup demonstrated that the Hca-P were transfectanted by the pcDNA3.1- Annexin A7 stably; Western-blotting indicated when we compared the optical density of Annexin A7 with the optical density of GAPDH, we found that the value in transfected Hca-P was significantly higher than that in normal Hca-P (0.43544±0.0112 vs 0.45957±0.0065, P<0.05).②The sequencing results confirmed that the sequences of three shRNAs were correct. Compared with nomal Hca-F cells, the Annexin A7 protein expression was down-regulated significantly in those cells which were transfected stably with the shRNA (P<0.05).③The motility abilities of Hca-P cells were improved because of the up-regulated Annexin A7 expression in it; After the repression of Annexin A7 expression in Hca-F cell clones, the motility and invasiveness abilities of Hca-F cells were obviously inhibited, the apoptosis rate of Hca-F cells were increased and cell viability was lowered. Conclusion: The motility and invasiveness abilities of Hca-F were obviously inhibited, the apoptosis rate were increased and cell viability were lowered due to down-regulated the Annexin A7 expression;②The motility abilities of Hca-P cells were improved because of up-regulated Annexin A7 expression in it.③These facts indicated that Annexin A7 overexpression positively correlated with metastatic potentials of mouse hepatocarcinoma cells, especially lymphatic system metastasis, and suggested that Annexin A7 could represent a new potential target for gene therapy.