The Mechanism Research On CD82 Small Extracellular Loop Inhibiting Cell Migration And Inducing Apoptosis

CD82,a member of transmembrane 4 superfamily(TM4SF)protiens,is coded by the tumor-suppressor gene KAI1 and plays an important function in cell aggregation,adhesion,migration and regulation.CD82 is widely expressed in normal tissues while expressed down or not in tumor tissues.A lot of researches have shown that CD82 inhibits the migration of tumor cells from a variety of organizations both in vitro and in vivo,and is considered a broad spectrum of tumor metastasis suppressor.Now,the study of its mechanism of action is drawing more and more attention.The structure of CD82 contains three parts: extracellular region,transmembrane region and intracellular region.The extracellular region is made up of a small loop(SEL)and a large loop(LEL)and the transmembrane region has four hydrophobic peptides while the intracellular area including N-end,C-end and a cell inner-loop.The study of CD82 different domains has important theoretical significance to expound its molecular mechanism of inhibiting tumor metastasis.At present,most researches are focused on the function of CD82 extracellular region,which is concerned in CD82 polymerization and interaction with other membrane proteins.However,the study of the small loop in CD82 extracellular region is rare,leading to its function not yet clear.To understand the role of SEL in CD82 inhibiting tumor metastasis,we chemically synthetic the CD82-SEL amino acid sequence simulation peptide(CD82-SEL-P)and study its role in cell migration,proliferation and apoptosis.1.The influence of CD82-SEL-P on cell in vitro migration,adherent growth,and cell-cell adhesion.The scratch method and Boyden chamber culture method were carried out to observe the influence of CD82-SEL-P on migration,adherent growth,and cell-cell adhesion in vitro.Results:(1)CD82-SEL-P can significantly inhibit the tumor cell migration in vitro,and its inhibition rate is positively correlated with the concentration of CD82-SEL-P.(2)CD82-SEL-P can significantly inhibit adherent growth,which leads to cells gather mass of suspended growth.And the ability to inhibit cell adherent and treatment concentration were positively correlated.(3)CD82-SEL-P can promote the growth of suspension cells.Conclusion: CD82-SEL-P can inhibit adherent cell migration in vitro;CD82-SEL-P can inhibit the growth of adherent cells and leads to gather mass of suspended growth;CD82-SEL-P promotes the growth of suspended growth of cells.And the inhibition rate is positively correlated with the concentration.Taken together,CD82-SEL-P may have special independent function beyond CD82 or important structural domain related to the inhibition of metastasis.This conclusion is reported for the first time.2.The influence of CD82-SEL-P on tumor cell growth and proliferation.We used the cell fluorescence histochemistry and MTT method to detect the effect of CD82-SEL-P on different tumor cell growth and cellular morphology.Results:(1)The treatment of CD82-SEL-P can lead to the tumor cells morphological changes,cell shrinkage,wrapped slurry particles,cavitation and shedding from the plate.(2)CD82-SEL-P can suppress the growth and proliferation of various solid tumor cells.And its inhibition is depended on concentration.(3)CD82-SEL-P promotes the growth and proliferation of suspension cell.Conclusion: CD82-SEL-P has opposite effct on the growth of adherent cells and suspension cells.CD82-SEL-P inhibits the growth and proliferation of cultured adherent cells in vitro but promotes the growth of suspension cell proliferation.3.To explore the molecular mechanism of CD82-SEL-P by which inhibit cell migration,we synthesized biotin labeled CD82-SEL-P as a probe,and explored the potential target molecules(including membrane proteins and membrane lipid)of CD82-SEL-P by using Western blot.Results:(1)According to the results of the Western blot,biotin labeling CD82-SEL-P probe can recognize more than ten proteins.What the CD82-SEL-P recognized are membrane proteins rather than cytoplasm protein.(2)Biotin labeled CD82-SEL-P can be combined into total lipids packs by silica gel ball,illustrating the targets that CD82-SEL-P identified and combined contain lipid composition.Conclusion: What the CD82-SEL-P identified and combines is the specific structure of oligosaccharide chains,its target is not a specific protein or lipid molecules,but multiple molecules(including glycoproteins and glycolipids).4.CD82-SEL-P induces SW620 cells and MDA-MB-231 tumor cell apoptosis and mechanism research.In this part,we further observed the influence of CD82-SEL-P in inducing SW620 cells and the MDA-MB-231 tumor cell apoptosis,and studied its mechanism.Results:(1)According to the results of the cellular network stations,CD82-SEL-P can induce SW620 cell and MDA-MB-231 tumor cell apoptosis.(2)As Western Blot show,CD82-SEL-P treatment can increase Caspase3 and Caspase8 expression in SW620 and MDA-MB-231 cells.(3)CD82-SEL-P induced SW620 cell apoptosis.Meanwhile,the mitochondrial membrane declined,illustrating CD82-SEL-P inducing SW620 cells apoptosis may be involved in mitochondrial apoptotic pathways.(4)CD82-SEL-P induced SW620 cells apoptosis,at the same time,the expression of Caspase12 and GRP78 increased,prompting CD82-SEL-P inducing MDA-MB-231 cells apoptosis may be involved in endoplasmic reticulum apoptosis induction.Conclusion:(1)CD82-SEL-P can induce apoptosis in SW620 cells and MDA-MB-231.(2)CD82-SEL-P induced different tumor apoptosis by different apoptotic pathways.CD82-SEL-P induced SW620 cell apoptosis mainly through mitochondrial apoptotic pathway,while the MDA-MB-231 cells mainly through endoplasmic reticulum apoptotic pathway.