The Influence Of Human Selenoprotein Selk On Cancer Cells And The Construction Of Its Recombinant Adenovirus Vector

Selenoprotein SelK is one of the selenoproteins in mammalian bodies,which is located on endoplasmic reticulum and Golgi bodies.It has very important roles in calcium flux and macrophage activation.The tumor cells transfected with human selenoproteins SelK showed signs of apoptosis.Since the imbalance of intracellular calcium homeostasis is one of the key factors to induce cell apoptosis,and the change of Ca²⁺concentration also has effects on cell migration,the effects of human selenoproteins SelK on cytosolic calcium concentration,cell apoptosis,adhesion and migration of different tumor cells were studied.And the influences were compared with that on human embryonic kidney cells.First,the effect on human tumor cells of the recombinant plasmids containing full-length sequence SelK and truncated sequence were studied by liposome transfecting theses plasmids into human gastric cancer cell BGC-823 and hepatoma cell HepG-2,and human embryonic kidney cell HEK-293 was used as control.Cell viability was examined by MTT assay 48h after was showed that the viability of BGC-823 and HepG-2 cells overexpressing C-terminal truncated sequence of SelK was not significantly inhibited?P>0.05?compared with that of non-transfected cells and that transfected with empty plasmid.But the viability of BGC-823 and HepG-2cells overexpressing SelK full sequence of were significantly reduced?P<0.05?.The viability of the overexpression of complete SelK or truncated SelK had no obvious change in human embryonic kidney 293 cells.Fluo-3 AM was then used as a Ca²⁺probe and confocal microscopy analysis was used to compare the changes of cytosolic free Ca²⁺level of these cells.It was showed that,in the three cells,the overexpression of complete SelK significantly increases the cytosolic free Ca²⁺level and the difference with the control group was significant.The cytosolic free Ca²⁺level of BGC-823and HepG-2 cells after the overexpression of complete SelK increased 4 times and 3 times compared with that of the overexpression of C-terminal truncated SelK and the variation was very significant?P<0.01?.Next,the apoptotic ratio of the BGC-823,HepG-2 and normal HEK-293 cells induced by pDsRed2-C1-SelK-secis transfection was examined by flow cytometry following FITC–labeled annexin V staining.It was showed that the over expression of complete SelK could induce BGC-823 cells apoptosis and the ratio of apoptotic cells reached 32.7%.The differences were statistically significant compared with that of C-terminal truncated SelK and that of empty plasmid?P<0.05?.The apoptosis ratio of HEK-293 which overexpressing complete SelK was not significantly different compared with cells transfected with empty vector plasmid and overexpressing truncated Selk?P>0.05?.Finally,The effect of human selenoprotein SelK on cell matrix adhesive and cell migrational ability of BGC-823 cells,HepG-2 and HEK-293 cells was detected by matrigel adhesion assay and transwell chamber assay.It was shown that the matrix adhesive ability and cell migrational ability of BGC-823 cells which overexpressed truncated SelK was not significant changed compared with non-transfected cells and the cells transfected with empty vector.But the matrix adhesive and cell migrational ability of BGC-823 cells which overexpressed complete SelK decreased significantly?P<0.05?and was very significantly?P<0.01?,respectively.The research results in HepG-2 cells very similar to that in BGC-823 cells.But in normal HEK-283 cells,the effect of the overexpression of complete SelK and the truncated form had no significant difference on cell adhesion and migration.In order to improving the transfection efficiency of SelK expression and eliminating the influence of liposome reagent or other factors during liposome transfection,the recombinant adenoviral vector of mouse selenoprotein mSelK,Ad-mSelK-secis,was constructed for further researching.Since the protein sequence of mouse selenoprotein mSelK and human selenoprotein SelK are basically the same,the mouse recombinant adenovirus vector of selenoprotein mSelK could be verified by the overexpression in human cancer cells and used for the researches on the molecular functions of human SelK.When Ad-mSelK-secis was transfected into HepG-2 cell,the inhibition on cell viability reached very significant level?P<0.01?.The inhibitive ability was two times of that transfected by plasmids containing full-length SelK and the cytosolic Ca²⁺level was further increased was suggested that the recombinant adenovirus vector Ad-mSelK-secis has significantly improved the transfection efficiency than the plasmids containing full-length SelK and may further be used to study the effect of SelK on human cancer cells and other aspects.In summary,by transfecting human recombinant plasmids containing SelK-secis into tumor cells BGC-823 and HepG-2 we confirmed that human selenoprotein SelK could promote the elvation of cytosolic Ca²⁺levels of tumor cells and induce the cell apoptosis,inhibit matrix adhesion and migration of tumor cells.Although SelK could also increase the cytosolic Ca²⁺level of human embryonic cells,the ratio of appotic cells,the adhensive and migrational ability of the cells were not significantly influenced.The recombinant adenovirus vector Ad-mSelK-secis greatly improved the transfection efficiency of the protein and could be used for further illuminating the biological functions of SelK,and also provided a new way for the tumor treatment.