Study On The Correlation Between The Expression And Localization Of CD82 And Integrins α6,β4,β1 In Breast Cancer

Background:According to the Latest Global Cancer Data,there is a significant increase in the number of newly diagnosed cases of breast cancer in 2020,which requires urgent attention.25%～28% of patients diagnosed with breast cancer for the first time had metastasis,and the cure rate decreased significantly due to metastasis.Therefore,early screening of breast cancer and targeted diagnosis and treatment of metastatic breast cancer are particularly important.As a wide range of tumor suppressor,down-regulation of CD82 can lead to breast cancer metastasis.CD82 interacts with integrin,chemokine,epidermal growth factor receptor and other tetraspanins to form tetraspanin-enriched membrane microdomains(TEMs),plays a key role in cell motility,adhesion and signal transduction.Integrin is involved in the adhesion of cells to extracellular matrix.ITGA6,ITGB4,and ITGB1 are all members of laminin binding subfamily,which have the ability to promote tumor progression and metastasis.As an exosome marker,tetraspanins are closely related to exosome production and intercellular communication.There is a relationship between the expression of integrin in exosome and organ targeted metastasis.Our previous study found that the expression of CD82 in the exosome of breast cancer was significantly higher than that in patients with benign breast diseases and healthy subjects,and negatively correlated with the expression of CD82 in tissues.Then the expression and localization of CD82 and integrin in breast cancer and their relationship in exosomes need to be further explored.Objective:To explore the relationship between CD82 and the expression and location of integrins ITGA6,ITGB4,and ITGB1 in breast cancer;analyze the correlation between CD82 and the expression of integrins ITGA6,ITGB4,and ITGB1 in breast cancer exosomes.Method:1.Detection of the expression of CD82,ITGA6,ITGB4,and ITGB1 in four kinds of breast cancer cells by Western Blot.2.Screening of plasmids with the most obvious effect of interfering with the expression of CD82 by Western Blot and immunofluorescence.3.Detection of interaction between CD82 and ITGA6,ITGB4,ITGB1 in breast cancer cells by protein co-immunoprecipitation(Co-IP).4.The differences of localization and expression of ITGA6,ITGB4,and ITGB1 among control group,shRNA-CD82 group and wild type group were detected by Western Blot and cellular immunofluorescence.5.Exosome markers,CD9 and tumor susceptibility gene(TSG101),were detected by Western Blot to identify exosome.6.Detection of the expression of CD82,ITGA6,ITGB4,and ITGB1 in serum exosome of breast cancer,benign breast diseases and healthy subjects by Western Blot.Results:1.The expression of CD82 in shRNA-CD82-788 group of breast cancer MDA-MB-231 cells was significantly weaker than that in control group(P<0.05),and the fluorescence signal intensity of CD82 in shRNA-CD82-788 group was lower than that in control group.2.CD82 can interact with ITGA6,ITGB4,and ITGB1 to form complexes in breast cancer MDA-MB-231 cells.3.The expression of total protein,ITGB4 and CD82 in MDA-MB-231 cells of breast cancer was negatively correlated,while the expression of ITGA6 was positively correlated with that of CD82(P<0.01).There was no significant difference in ITGB1 expression among control group,shRNA-CD82-788 group and wild type group(P>0.05).4.CD82,ITGB4,ITGB1 and ITGA6 were co-located in the cell membrane and cytoplasm,but there was no difference in the distribution of the three integrins among the control group,shRNA-CD82-788 group and wild type group.The fluorescence intensity of ITGB4 in the wild type group was weaker than that in the control group,and the fluorescence intensity of ITGA6 was stronger than that in the control group,and the fluorescence intensity of ITGB4 was enhanced and that of ITGA6 decreased in shRNACD82-788 group.There was no significant difference in ITGB1 fluorescence intensity among control group,shRNA-CD82-788 group and wild type group.5.The relative expression of CD82 in serum exosome of patients with breast cancer was higher than that of healthy people and patients with benign breast disease,while the relative expression of ITGA6 in patients with breast cancer was significantly lower than that of healthy people and patients with benign breast disease(P<0.05).There was no significant difference in the relative expression of ITGB1 in serum exosome of patients with breast cancer,patients with benign breast disease and healthy people(P>0.05).The expression of ITGB4 in serum exosome was not detected in the experimental samples.Conclusion:1.CD82 interacts with ITGA6,ITGB4,and ITGB1 in breast cancer MDA-MB-231 cells,and co-locates in the cell membrane and cytoplasm.2.CD82 can regulate the expression of integrin in breast cancer MDA-MB-231 cells,which is negatively correlated with ITGB4 and positively correlated with ITGA6.3.The relative expression of ITGA6 in serum exosome of breast cancer patients was lower than that of benign breast disease and healthy subjects,and it was negatively correlated with the expression of CD82 in exosome.