ShRNA Inhibition Of Annexin A7Inhibits Proliferation And Migration In Mouse Hepatocacinoma Hca-P Cells

Background: Among malignant tumors, primary liver cancer is one of the mostmalignant tumors with the highest death rate. Radical surgical resection is the preferredmethod of treatment, but after five year relapse rate and metastasic rate is still high. Oneof the most important biological characteristics of cancer is metastasis. Liver cancerbelong to the malignant tumors of epithelial origin which has high metastatic potentialand metastasizes early to regional lymph nodes. Such regional lymph node metastasisare the most important influencing factors resulting in poor prognosis and highmortality in patients with malignant tumors. Therefore, the study of liver cancer lymphnode metastasis-related genes will be of potential benefit to decrease the relapse anddeath rates of cancer patients and contribute new ideas to clinical treatment.Objective:1. pGPU6/GFP/Neo-shRNA-Annexin A7and pGPU6/GFP/Neo-shRNA-NC expression vectors were constructed and stably transfected into Hca-Pcells.2. CCK8was used to study the proliferation ability of the cells and transwellchamber as the cell migration assay to investigate differences in cell migration ability.Method:1. pGPU6/GFP/Neo-shRNA-Annexin A7and pGPU6/GFP/Neo-shRNA-NC expression vectors were constructed and stably transfected into Hca-Pcells to obtained PAnxa7down regulatedcells and PAnxa7no relationship consequencecells respectively.Hca-P cells and PAnxa7no relationship consequencecells were used as negative controls.2. Realtime qRT-PCR and Western blot were used to verify the down regulation of AnnexinA7at the mRNA level and the protein level respectively. CCK8was used to study theproliferation ability of the cells and transwell chamber as the cell migration assay toinvestigate differences in cell migration ability.Results:1. Automatic DNA sequencing confirmed expression vector sequencingresults was the same as the designed sequence meaning that the expression vector wassuccessfully constructed.24hours after transfection, the transfection efficiency was confirmed by observing the green fluorescent protein expression in the transfected cellsunder inverted fluorescence microscope.2. Real time qRT-PCR and Western blotresults verified significant down regulation of Annexin A7at both the mRNA andprotein levels. The CCK8experiments showed PAnxa7down regulatedcells’ growth rate wassignificantly lower than the Hca-P cells and PAnxa7no relationship consequencecells, indicatingthat Annexin A7down regulation inhibits cell proliferation. Transwell chamberexperiments showed that the number of PAnxa7down regulatedcells that migrated through theTranswell chamber was significantly reduced, also indicating that the Annexin A7downregulation decreases cell migration ability.Conclusion:1. the target gene was successfully built into pGPU6/GFP/Neo-shRNA expression vector, stable transfection achieved.2. Both Real time qRT-PCR andwestern blot confirmed successful down regulation of the gene. The significant decreasein cell proliferation with the CCK8experiment and also the marked decrease in cellmigration with the transwell experiment indicate that Anxa7might be a significantoncogene and could be a therapeutic target for further research in to the mechanisms ofliver cancer lymphatic metastasis.