Quantitative Research Into The Subcellular Positioning Of Anxa7, Jnk And Gsn In Tumor Cells

Objective:Hepatocellular carcinoma （HCC） is a major human killer. Liver cancerresearch has made great progress over the years and the treatment also graduallyimproved, but the question of survival of patients is still very grim. Liver cancermetastasis is the cause of the short survival time of patients with hepatocellularcarcinoma, therefore understanding the mechanisms and factors influencinghepatocellular lymphatic metastasis is necessary for the improvement in clinicaltreatment of liver cancer and prolonging the survival time of patients.Hca-F cells and Hca-P cells are mouse ascites hepatoma cell lines from the sameparents, highly homologous with similar genetic backgrounds,but with differentlymphatic metastatic potential of. Hca-F cells has high lymphatic metastatic potentialwith lymph node metastasis rate>70%; whilst Hca-P cells has lower lymphaticmetastatic potential with lymph node metastatic rate <30%. Our laboratory in a previousstudy applied suppression subtractive hybridization, gene chip technology andquantitative proteomics technology to find genes differentially expressed in Hca-F cellsand Hca-P cells. membrane associated proteins A7（Anxa7）, c-Jun N-terminal kinase（Jnk）, gelsolin （Gsn） genes showed high expression in the Hca-F cells, suggesting thatthey are related to liver cancer lymph node metastasis.Anxa7is a member of the annexin family of calcium-dependent proteins, it canpromote membrane binding, aggregation and fusion, inhibition of phospholipase Aactivity in the cells, and to participate in the activities of the cell phospholipids and thecytoskeleton, anticoagulation, regulation of membrane receptor function, promote cellsecretion, and plays a very important role in cell signal transduction process. Jnk is amitogen-activated protein kinase （MAPK） superfamily members, one of the veryimportant signal transduction pathways. It is able to participate in a series ofphysiological processes of cell differentiation and apoptosis. Gelsolin is one of the members of the gelsolin superfamily, it is a very important actin binding protein. InCalcium-dependent manner, it can control the actin protein structure, the reorganizationof actin, and able to participate in cell motility, and programmed cell death. Gsn alsocan be used as the auxiliary of the transcriptional activator protein, at the same time canpromote the transcriptional activity of the androgen receptor.Methods: In this study, subcellular proteomics approach was used. Hca-F cells,Hca-P cells, Hca-F stably transfected with PG-CMV-EGFP-Kan/neo-Annexin A7expression vector of (F_{Anxa7上调}cell), Hca-P cell lines stably transfected with shRNAexpression vector pSilencer3.1-Hca-P P_{Anxa7下调}cell) were fractionated into four cellularcompartments. Western Blot method was used to identify the differences in thesubcellular locations and function of Anxa7, Jnk, and Gsn to lay a solid foundation forfuture research.Results:Anxa7: Anxa7can be shown in Cytosolic fraction, Membrane/organelle proteinfraction, and Cytoskeletal fraction in F_Anxa7upcell and P_Anxa7downcell. The expression ofAnxa7in Cytosolic fraction, Membrane/organelle protein fraction, and Cytoskeletalfraction in F_Anxa7upcell is higher than in Hca-F（P <0.05）, and in P_Anxa7downcell is lowerthan Hca-P（P <0.05）. Jnk can be shown in Nucleic protein fraction and Cytoskeletalfraction in F_Anxa7upcell and P_Anxa7downcell. Gsn can be shown in Cytosolic fraction,Membrane/organelle protein fraction, and Cytoskeletal fraction in F_Anxa7upcell, But onlyin Cytosolic fraction and Cytoskeletal fraction in P_Anxa7downcell.Conclusion:1. In F_Anxa7upcell and P_Anxa7downcell, Anxa7can be shown in Cytosolic fraction,Membrane/organelle protein fraction, and Cytoskeletal fraction. Jnk can be shown inNucleic protein fraction and Cytoskeletal fraction. Gsn can be shown in Cytosolicfraction, Membrane/organelle protein fraction, and Cytoskeletal fraction in F_{Anxa7上调}cell, But only in Cytosolic fraction and Cytoskeletal fraction in P_Anxa7downcell.2. The expression of Anxa7in Cytosolic fraction, Membrane/organelle proteinfraction, and Cytoskeletal fraction in F_Anxa7upcell is higher than in Hca-F（P <0.05）, andin P_Anxa7downcell is lower than Hca-P（P <0.05）.