Effect Of CD82 Palmitoylation Modification On Proliferation And Metastasis Of Triple N Egative Breast Cancer MDA-MB-231 Cells

Objective:The broad-spectrum metastasis suppressor gene CD82/KAI1,which is an important member of the four-transmembrane protein family,suggests that CD82 palmitoylation modification is important for CD82 expression on cell membranes and localization in the microdomains of four transmembrane proteins.However,the role of CD82 palmitoylation modification in maintaining CD82 function has not been fully elucidated.The effect of CD82 palmitoylation modification on signal transduction pathways involved in the proliferation and metastasis of triple negative breast cancer MDA-MB-231 cells were observed by three different palmitoylation site mutations closely related to CD82 function and the effect of proliferation and migration ability in vitro.Methods:First,the palmitoylation mutant lentivirus was constructed to construct CD82 mutants with different palmitoylation site mutations(including Cys5,Cys74,Cys83 single point mutations,and Cys5/Cys74,Cys5/Cys83,Cys74/Cys83,Cys5/Cys74/Cys83 multiple mutants with different combinations),with lentiviral vector alone as the negative control,blank group,overexpressing CD82 as CD82-WT group.and different palmitoylation site mutants were labeled as CD82-C5?CD82-C74?CD82-C83?CD82-C5.C74?CD82-C5.C83?CD82-C83.C74?CD82-C5.C74.C83.Secondly,Q-PCR and Western Blot were used to detect the expression of CD82 in breast cancer cells.Then,the effects of CD82 palmitoylation mutation on the proliferation and migration of breast cancer cells were detected by cell growth assay(CCK 8),scratch method and Transwell chamber.Finally,Western blot was used to detect the effects of CD82 palmitoylation mutationon growth factor receptor activity and signaling pathway activity associated with tumor proliferation and metastasis.Results:1.The CD82 mutants with different palmitoylation site mutations were detected by sequencing method.The results showed that all the CD82 different palmitoylation site mutants were correctly sequenced,indicating that the CD82 palmitoylation mutant was successfully constructed2.Western Blot and Q-PCR were used to detect the expression of CD82 in breast cancer cells.The results suggested that make no significant difference in the expression of CD 82 palmitoylation mutant relative to wild type CD 82 after transfection of CD 82 palmitoylation mutant3.The effect of CD 82 palmitoylation mutation on the migration of breast cancer cells was detected by scratch method and Transwell method.The results indicate that Cys 83 palmitoylation mutation of CD 82 or simultaneous mutation of Cys 5 and Cys 74 palmitoylation can promote MDA-MB-231 cell migration4.CCK8 detects the effect of CD 82 palmitoylation mutation on the growth of breast cancer cells.The results suggested that the CD82 palmitoylation cysteine residue 5,74,83 mutations can promote the proliferation of breast cancer cells5.Western Blot was used to detect the effect of CD 82 palmitoylation mutation on epidermal growth factor receptor(EGFR)and hepatocyte growth factor receptor(HGFR)in breast cancer cells.The results indicate that the Cys 83 palmitoylation mutation of CD 82 can activate the phosphorylation of the EGFR tyrosine 1173;the Cys 83 palmitoylation mutation of CD 82 and the simultaneous mutation of Cys 5 and Cys 74 palmitoyl can activate EGFR tyrosine 845 phosphorylation.Cys 5,Cys74,Cys 83 palmitoylation mutations of CD 82 and simultaneous palmitoylation of Cys 5 and Cys 74 can activate HGFR tyrosine 1313 phosphorylation;CD 82 Cys 5 and Cys 74 phosphorylation simultaneous mutation can activate HGFR tyrosine 1365 phosphorylation6.Western Blot assay was used to study the effect of CD 82 palmitoylation mutation on the activity of major signal transduction pathways in breast cancer cells related to the proliferation and migration of tumor cells.The results showed that Cys 83 palmitoylation mutation of CD 82 and simultaneous mutation of Cvs 5 and Cys 74 palmitoylation can activate PI3K/Akt signal pathway;Cys 5,Cys 74 and Cys 83 palmitoylation mutations of CD 82 can activate MAPK signal Pathway;CD 82 palmitoylation mutation does not affect the activity of the PLC y signal pathway.Conclusion:Different palmitoylation site mutations have different effects on the proliferation and metastasis of MDA-MB-23 1 breast cancer cells.1.The Cys 83 palmitoylation mutation of CD 82 and the simultaneous palmitoylation of Cys 5,Cys74 canrespectivelyactivate the EGFR tyrosine 1173 phosphorylation and 845 phosphorylation,and further respectivelyactivate phosphorylation of Akt threonine 308 and Akt serine 473 in the PI3K/Akt signaling pathway,thereby promoting MDA-MB-23 1 Cell migration.2.The Cys 5,Cys 74,Cys 83 palmitoylation mutationof CD 82 and the simultaneous palmitoylation of Cys 5 and Cys74 can respectivelyactivate HGFR 1313 phosphorylation and/or 1365 phosphorylation,further respectivelyactivateof phosphorylation in the MAPK signaling pathway,thereby promoting MDA-MB-231 cell proliferation.3.The CD82 palmitoylation mutation does not affect the activity of the PLCy signaling pathway.