Study On The Mechanism Of N-glycosylation Of CD82 In Regulating The Metastasis Of Colon Cancer

Background: Glycosylation of proteins is the most common post-translational modification.There are mainly four types of glycosylation: N-connection,O-connection,GPI anchoring and C-connection.Studies have shown that N-glycosylated glycoproteins play a role in the recognition process between cells and cells,cells and matrix.Therefore,the mechanism of N-glycosylation of CD82 in the recognition and adhesion of cells and the invasion and migration of tumor cells has become the main starting point of this study.It is known that CD82 is transmembrane glycoprotein,there are three N-glycosylated residues in its extracellular domain,which at Asn129,Asn157 and Asn198 residues.Previous studies have suggested that N-carbohydrate chain is necessary for CD82 to inhibit tumor metastasis.Therefore,we speculate that N-carbohydrate chain may be an important target for interaction and recognition between CD82 and other molecules.It is an important structure to participate in the correct distribution of CD82 in the membrane microdomains or the formation of the tetraspan transmembrane protein complex.Due to the role of N-glycosylation of CD82 in the inhibition of tumor metastasis,it could be used as a tumor marker for predicting the prognosis of patients with malignant colon cancer,and provides a theoretical basis for clinical treatment of tumor metastasis.Objective: In this study,we observed the changes of cell biological behaviors of N-glycosylation mutants of CD82 at different residues of N-glycosylation in the process of colon cancer metastasis,this study preliminarily clarified the molecular mechanism of its regulation of metastasis of SW620 cells,and verified it in vivo test of animals,providing a new direction for the clinical diagnosis and treatment of colon cancer.Methods: 1.N-glycosylation mutants of CD82 were constructed and transfected to SW620 cells.The expression level was detected by q PCR and Western Blot.2.The effect of different N-glycosylation mutants of CD82 on cell movement,migration andadhesive capacity were detected by cell wound scratch assay,transwell assay and cell adhesion experiments.3.The expression and phosphorylation level of the vital molecular of WNT signaling pathway including Beta-catenin,GSK3? and Vimentin(EMT marker)were detected to explore their metastasis.4.The pulmonary metastasis ability of SW620 cells transfected with different N-glycosylation mutants of CD82 were studied by the model of nude mice.Results: CD82 mutants with different N-glycosylation residues,including Asn129/Asn157,Asn157/Asn198,Asn129/ Asn198 double residues mutations and Asn129/Asn157/Asn198 three residues mutants,were constructed successfully.Take the wild type SW620 cell group without transfection as the control group,through scratch test and transwell test,it was found that the mutants Asn129/Asn157,Asn157/Asn198 and Asn129/Asn157 /Asn198 of CD82 lost the function of inhibiting tumor metastasis.While compare with the control,mutation Asn129/Asn198 of CD82 still kept the ability to inhibit tumor metastasis as wild CD82(p<0.0001).Cell adhesion experiments showed that compare wtith the control,the Asn129/Asn198 mutants of CD82 still kept the ability to inhibit adhesive capacity(p<0.0001).To explore the effects of different N-glycosylation of CD82 on Wnt/?-catenin signaling pathway,downstream effector molecules in this pathway have been detected.The results showed the phosphorylation level of GSK3? was suppressed in SW620 cells transfected with Asn129 / Asn198 mutant of CD82 comparing with the contro(p<0.01),which caused accumulation of phosphorylation level of ?-catenin.Then the Wnt/?-catenin signaling pathway was blocked by degradation of high level phosphorylated ?-catenin(p < 0.01),which inhibited cell transformation of EMT and enhanced SW620 cells adhesion.Moreover,the expression of Vimentin decreased in SW620 cells transfected with Asn129 / Asn198 mutant of CD82 supported the conclusion(p<0.05).BLAC/c-Nude mice were used to establish animal models by tail vein injection of SW620 cells transfected with different N-glycosylation mutants of CD82.The results showed that the number of tumor metastases were significantly lower in group of SW620 cells transfected with mutant Asn129/Asn198 and wild CD82 than those of other mutants.Conclusion: N-glycosylation at Asn157 residue of CD82 plays an important role in maintaining the function of inhibiting tumor metastasis.It can inhibit the movement and adhesion of colon cancer effectively,and may participate in the process of blocking the metastasis of colon cancer through the Wnt/?-catenin signaling pathway.The research indicates that the N-glycosylation of CD82 can be used as a new tumor metastasis marker.It provides a new idea for diagnosis,treatment and prognosis of cancer patients.