The Expression Of ANXA7and Co-associated Protein ALG-2, SODD In Tumor Cells And Effection On Proliferation, Migration, Invasion In Vitro

Background:Uncontrolled cell proliferation and abnormal apoptosis, that is themalfunctioning of the mechanism of regulating the balance between cell proliferationand death in the body, is an important cause of the occurrence and development ofmalignant tumors. Primary liver cancer, a kind of malignant tumor originating fromthe epithelium, is one of the common malignant tumors in China and is also one of thetumors with the highest degree of malignancy. The current preferred method oftreatment is radical surgery. However, the five-year survival rate and relapse ratestubbornly remained on the top. Biological characteristics of malignant tumorsmanifest as unlimited proliferation, metastasis and invasion, among which thepotential of migration and metastasis are the most important signs. The metastaticways of liver cancer are divided into hematogenous metastasis, lymphatic metastasisand implantation metastasis. Among them, the lymphatic metastasis, which is also themost important factor of poor prognosis and high mortality of patients, occurs in theearly stage. It is well-known that although proteins are the executors of cell function,they are not able to play a role independently but combined with other proteins orsubunits for mutual regulation and joint action. Therefore, the research on therelationship between proteins will be relevant to revealing the mechanism of action ofproteins, and be more helpful to reduce the relapse rate and mortality rate in patientswith malignant tumors, thus providing a new treatment idea for clinical application. Objective:To use immune precipitation and cell immunofluorescence in eukaryotic cells todemonstrate that SODD （Suppressor Of Death Domains） and ALG-2（apoptosis-linked gene-2protein） are the interacting proteins of ANXA7（AnnexinA7）.Down-regulating the ANXA7gene expression levels of the mouse hepatomaHca-P cells and investigate the influence of ANXA7gene and protein on ALG-2andSODD as well as their protein expression levels.Methods:Use immune precipitation assay to determine whether or not SODD and ALG-2areANXA7-interacting proteins in eukaryotic cells. Use cell immunofluorescenceassay to determineALG-2and SODD are co-located ANXA7.pGPU6/GFP/Neo-shRNA-ANXA7and pGPU6/GFP/Neo-shRNA-NC expressionvectors were constructed and stably transfected into Hca-P cells to obtain PAnxa7–Downcell and PAnxa7–NGcell respectively. Hca-P cells and PAnxa7–NGcell were used ascontrol groups. Real-time PCR and Western blot were used to verify ANXA7proteinexpression after down-regulating, and its effect on SODD and ALG-2, both at mRNAand protein levels. Transwell experiment was used to observe the capacity of invasionand migration after down-regulating ANXA7protein.CCK-8assay were measured in the0hour,24hours,48hours,72hours of optical density value, the expression level of ANXA7、ALG-2and SODD were found to have correlation with the proliferation ability.Results:The immune co precipitation results: In Hca-P cells, SODD and ALG-2proteinbands appeared in the group to which ANXA7antibody was added, but did notappear in the group to which Normal IgG was added. so ANXA7, SODD and ALG-2are Interacting proteins. The cell immunofluorescence results: In Hca-P cell, ALG-2are co-located ANXA7nearly cytomembrane; SODD are co-located ANXA7nearly cytomembrane in cytolymph. At the mRNA level, ANXA7gene expression fell to21%ofthe original genetic level in PAnxa7–Downcell in contrast to PAnxa7–NGcell; ALG-2geneexpression fell to16%of the original genetic level in PAnxa7–Downcell in contrast toPAnxa7–NGcell cell, and SODD gene expression also fell to10%of the original geneticlevel in PAnxa7–Downcell in contrast to PAnxa7–NGcell. At the protein level, ANXA7protein expression fell to23%of the original genetic level in PAnxa7–Downcell incontrast to PAnxa7–NGcell; ALG-2protein expression fell to21%of the original geneticlevel in PAnxa7–Downcell in contrast to PAnxa7–NGcell; and SODD protein expression fellto28%of the original genetic level in PAnxa7–Downcell in contrast to PAnxa7–NGcell.Transwell experiment shows The number of PAnxa7–Downcell that passed through thefilter was lower than Hca-P cells and PAnxa7–NGcell. No significant differences wereobserved in the migration and invasion abilities between PAnxa7–NGcell and Hca-Pcells.CCK-8assay were measured in the0hour,24hours,48hours,72hours ofoptical density value. It shows that the proliferation abilities of PAnxa7–Downcell waslower than Hca-P cells and PANXA7-controlcell.Conclusion:In Hca-P cell, ANXA7, SODD and ALG-2are Interacting proteins; The cellimmunofluorescence results: In Hca-P cells, ALG-2are co-located ANXA7nearlycytomembrane; SODD are co-located ANXA7nearly cytomembrane in cytolymph; Theexpressions of SODD and ALG-2decreased as a result a7down regulation.Down-regulating the expression of ANXA7could inhibit migration、proliferation andinvasion abilities of Hca-P cells. This biological activity might be due to the jointinteractions among ANXA7, SODD and ALG-2.