The Structural Basic Research For Inhibition Of Tumor Cell Migration In Vitro And Metastasis In Vivo By Mimic Peptide Of Amino Acid Sequence Of CD82 Extracellular Small Ring Domain

So far,the metastasis of tumor cells in vivo is still the main cause of death in cancer patients.Therefore,understanding the molecular mechanism of metastasis of tumor cells and related molecules is very important for controlling metastasis and improve survival rate in clinical.In recent years,one of the important research achievements in the field of tumor metastasis has been the discovery of a class of proteins that play a negative role in tumor metastasis,which is characterized by inhibiting the diffusion and metastasis of tumor cells in vivo.But it does not affect the formation and growth of primary tumor.Therefore,it is defined as tumor metastasis suppressor.Its encoded genes are called transfer suppressor genes(Metastasis suppresor gene,Msgs).NM23 was the first metastasis suppressor gene discovered in1988.So far,more than 20 metastasis suppressor genes have been identified.The functions of different metastasis suppressor genes often cross each other.A metastasis inhibitor can also block metastasis at multiple steps of metastasis.Metastasis suppressor genes are often down-regulated or not expressed in metastatic tumor cells.Restoring its expression or function can inhibit the migration of tumor cells in vitro and metastasis in vivo.Therefore,the research of tumor metastasis suppressor and the treatment strategy of targeted metastasis suppressor have become the frontier in the field of tumor metastasis.Among these metastasis inhibitors,KAI1/CD 82 is one of the most detailed metastasis suppressor.KAI1/CD 82 is widely expressed in normal tissues.Its main biological functions are to regulate adhesion,migration,signal transduction and so on.In most tumor tissues,the expression of KAI1/CD82 is lost or down-regulated.KAI1/CD82 was first identified as a metastasis suppressor in prostate cancer by gene screening.A large number of subsequent studies have shown that KAI1/CD82 plays a role in metastasis inhibition in different tumors.KAI1/CD82 has been established as a broad-spectrum metastasis suppressor.KAI1/CD82 can inhibit tumor metastasis by multy of mechanisms.The process of tumor metastasis can be blocked in multiple steps,including promoting tumor cells homotypic adhesion and aggregation,inhibiting the exfoliation of tumor cells from primary tumors,and inhibiting the movement and invasion of tumor cells,inducing aging or dormancy of tumor cells and inhibiting the clonal proliferation of tumor cells in metastatic organs.Structurally,KAI1/CD82 is a member of the family of four transmembrane proteins,which contains four transmembrane domains,two extracellular rings(a small ring EC1 and a lager EC2),one small cytoplasmic ring and cytoplasmic N-terminal and C-terminal.The function of most of the domains of KAI1/CD82 has been clarified.The EC2 domain can mediate the interaction between protein and protein.It can also mediate homologous aggregation of CD82 molecules.Four transmembrane domains are involved in the assembly of(tetraspanin web).The cytoplasmic C-terminal is the key site to connect the important signal transduction molecules in the cell.However,so far,the study of extracellular ring of CD82 is rare and its function is not clear.The extracellular domain is known to be an important functional site of membrane proteins.It is an important domain that directly recognition and binds to extracellular matrix molecules such as laminin(Liminin),fibronectin(fibronectin),collagen and adhesion molecules and receptors on the flanking cells.Since the EC1 domain exists as a conserved structure in all members of the four-transmembrane protein family during long-term biological evolution,it should have certen specific functions.Therefore,our research focuses on the structure and function of extracellular ring of CD82.In previous studies,in order to understand the role of CD82 extracellular small ring domain in inhibiting tumor metastasis by CD82,we synthesized the polypeptide (CD82EC1-m P)according to the CD82 extracellular small ring domain amino acid sequence by chemical method,and studied its function in detail.The results showed that CD82EC1-m P had the function of CD82 intact molecule.Functional analysis in vitro showed that CD82EC1-m P could promote the Homologous adhesion and aggregation of tumor cells,inhibit the adhesion to extracellular matrix and migration of tumor cells.In vivo metastasis experiment,CD82EC1-m P can block lung metastasis of different types of tumor cells in allogeneic and xenogeneic transplanted mice.However,the mechanism of its action is not clear.Structure is the basis of function.In order to understand the molecular mechanism by which CD82EC1-m P inhibit tumor cell migration in vitro and metastasis in vivo,the main focus of this study is on the structural basis of the function of CD82EC1-m P.On the basis of the amino acid sequence of CD82 extracellular small rine domain,by adding polar,acidic,or basic amino acid residues,removing the two terminal amino acid residues and replace individual amino acid residues in the polypeptide chain to enhance its water solubility,changing the isoelectric point or charge properties of the polypeptide chain and changing the spatial conformation,a series of CD82EC1-m Ps derived peptides with different amino acids sequence were synthesized.The effects of different sequences of CD82EC1-m Ps on the ability of tumor cell migration in vitro and in vivo were compared and the mechanism was discussed.1.The effects of different sequences of CD82EC1-m Ps on the migration behavior of tumor cells in vitroBy cell culture and scratch method,the effects of different sequences of CD82EC1-m Ps on the homologous adhesion aggregation,adhesion growth and migration of tumor cells were observed to detect the effects of charge,spatial conformation changes and some amino acid residues on CD82EC1-m Ps function.1)CD82EC1-m P can be transformed into water-soluble by adding 4 alkaline ammonic acid residues K and R or acidic amino acid residues D and E on its N-terminal or C-terminal.2)The charge properties of CD82EC1-m Ps affect its functional activity.Negative charge inhibits its activity,while positive charge promotes its activity.3)The activity of CD82EC1-m Ps in inhibiting the migration and adhesion of tumor cells depends on the spatial conformation.4)Three amino acid residues at the N-terminal of CD82EC1-m P are not necessary for its function and can further shorten the peptide segment.2.The effects of different sequences of CD82EC1-m Ps on EMT and Wnt signaling pathway1)Changing the spatial conformation of CD82EC1-m P could reverse the down-regulation of Wnt signaling pathway and inhibiting of EMT induced by CD82EC1-m P.2)Adding acidic amino acid residues to CD82EC1-m P could reverse the down-regulation of Wnt signaling pathway and inhibition of EMT induced by CD82EC1-m P.3.The effects of CD82EC1-m Ps with different amino acid sequences on the metastasis of tumor cells in vivoTo detect the effects of charge properties and spatial conformation changes on tumor metastasis inhibition of CD82EC1-m P,Both LLC cell syngeneic mouse models and MDA-MB-231 cell xenograft mouse models have been established,respectively.The effects of the four of CD82EC1-m P(including N+4K,N+4E,V8P/L16 P and V8L/L16I)on the formation of lung metastases by different tumor cells in mice were studied.1.CD82EC1-m P can inhibit the formation of lung metastases in syngeneic mouse models and xenograft mouse models.2.The charge-bearing properties of amino acids affect the ability of metastasis inhibition of CD82EC1-m P.Negative charge reduced the metastasis inhibition of CD82EC1-m P.3)The inhibitory effect of CD82EC1-m P polypeptide on metastasis depends on normal spatial structure.CD824.Effects of changes in the spatial structure of CD82 extracellular small rine domain on the migration inhibition in Vitro and metastasis inhibition in vivo ofThe CD82 mutant plasmid CD82-M-V44P/L52 P was constructed by the replacement of 44 th and 52 th Amino acid residues of CD82 with proline(corresponding to the 8th and 16 th amino acid sequences of CD82EC1).After transfected with CD82-M-V44P/L52 P,the MDA-MB-231 cells were injected into nude mice via tail vein.The lung metastasis were detested.1.The change of the spatial structure of the extracellular small ring domain of CD82 could reverse the metastasis inhibition of CD82 in syngeneic and xenograft mouse models.2.The extracellular small ring domain is an important functional structure of CD82.