| Palladin is a widely expressed actin-associated protein.The function of palladin is to regulate actin organization and it localizes in focal adhesions, cell-cell junctions, dorsal and circular ruffles, and periodically along stress fibers where it colocalizes withα-actinin. It is required for the maintenance of normal stress fibers and focal adhesions in cultured cells. Actin microfilaments are one of the three major components of the cytoskeleton. All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, adhesion, migration, contraction and cell morphogenesis.In order to study the role of palladin on the cell migration and adhesion, we constructed the eukaryotic expression vector pcDNA3.1(+)-Flag-HPF to over-expresse palladin and knockdown vector pGenesil-1-palladin-shRNA to specifically knock-down palladin gene expression. In this study we utilized three lines of human tumor cells MCF7, Hela and HT1080. We examined weather palladin overexpression or knockdown could influence cell adhesion and migration in these three cell lines.In the migration assay, we transfected cells with vectors above and empty vectors (controls) to examined weather palladin had any effect on the cell migration by transwell and woune healing assay. The transwell analysis showed that the migration of three cell lines was increased when the palladin overexpressed, whereas the migration were decreasee following the reduction of the palladin content. When the palladin overexpressed, the migration percentage of MCF7, HeLa, and HT1080 cells raised by 39.3%,21.1% and 8.4%, respectively, compared with control. In contrary, when palladin expression was knockdowned by shRNA the migration percentage of these three cell lines decreased by 9.5%,25.4% and 35% respectively. The same results were obtained from wound healing assay. These results showed that palladin play an important role in the cell migration.To assess whether palladin has the effects on the cell adhesion, three types of cells mensioned above were used for the analysis of the abilities to attach to the extracellular matrix proteins such as fibronectin andβ-amyloid protein (Aβ42) after these cells were transfected with the same pcDNA3.1(+)-Flag-HPF and pGenesil-1-palladin-shRNA vectors. The results showed that the overexpression of palladin increase the cell adhesion, but the palladin-deficient cells showed the consistently decreased adhesion compared to controls. As the results, when the palladin overexpressed, the adhesion percentage of MCF7, HeLa, and HT1080 cells raised by 33.8%,18% and 9.4% respectively, in contrary, when the palladin low-expressed by shRNA the adheison percentage of these three cell lines decreased by 11.5%,20% and 26.3% respectively. These data clearly demonstrate that palladin might play a crucial role in cell adhesion on the extracellular matrix.On the other hand, we performed western blot assay to detecte the endogenic palladin in the the adhereing and suspending cells. The results showed that the level of palladin was much higher in the adhereing cells. Moreover , when the cell adhesiveness on the matrix coating with extra-cellular protein such as FN was higher than that on the uncoated-matrix. These results indicated a critical role of palladin in the cell adhesion and migration.The actin polymerization and the filament actin stability have been proposed to be the keys regulation enrolled by tumor cells to become metastasis. According to the references and the results obtained above, palladin plays the important roles in actin cytoskeleton organization, cell motility and cell adhesion in the axonal extension in the nervous system development. In conclusion, we provide the insight into the role of palladin in cancer metastasis resulted from its actin organization and cell motility. Further studies should be required to confirm whether palladin or/and other regulators represent the novel targets for therapeutic interventions in human cancer.
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