Effects Of CD82 Palmitoyl Modification On Apoptosis Of Breast Cancer Cells And Its Molecular Mechanism

Background:Breast cancer is the highest incidence of cancer in our country,according to data published by the National Cancer Center in 2018.In our country,about 27,000 new cases of breast cancer each year,the number of deaths is about 66,000,and the most important cause of death in breast cancer patients is the distant metastasis of the tumor cells.With the development of the medical technology,the treatment plan of the breast cancer is continuously improved,but the treatment effect of the breast cancer which has already occurred in the distant metastasis is still not ideal.So it is expected that the metastatic mechanism of breast cancer is expected to improve the prognosis of patients.In the study of the tumor metastasis mechanism,the transfer inhibition factor was found to control the metastasis of the breast cancer.Guiding clinical treatment provides a new way of thinking.CD82 is a membrane glycoprotein encoded by the suppressor gene KAI1 and belongs to the family of the four transmembrane proteins.It is widely expressed in the normal tissue cells of the body,but it is a broad-spectrum tumor metastasis suppressor in various tumor tissue expression or deletion^[1-2].In recent years,it has been found that the CD82 palmitate modification can affect the formation of a four-transmembrane protein network,regulate the positioning of the protein on the surface of the cell membrane,inhibit the cell migration mediated by the integrin,and may affect the cell apoptosis process.However,that present study still does not fully state how the CD82-palmitate modification is involved in the regulation of this process and its molecular mechanism.Objective:By mutation of three different palmitoyl sites closely related to the function of CD82 in MDA-MB-157,MCF-7 and SKBR-3 cells of breast cancer,the expression and proliferation of CD82 palmitoylation on different breast cancer cells were observed.The effect of migration and apoptosis,and its molecular mechanism.Method:Single point mutation and multi-point combination mutation of Cys5,Cys74 and Cys83sites of CD82 palmitylation in breast cancer cells were used to construct CD82palmitoyl mutants（i.e.,Cys5,Cys74,Cys83,and Cys5+Cys74,Cys5+Cys83,Cys74+Cys83,Cys5+Cys74+Cys83）.The lentivirus plasmid packaging of CD82 mutant was completed by Shanghai Geen pharma Co.,Ltd.,China.The synthetic lentivirus plasmid was transfected into M respectively.There are three types of breast cancer cells,DA-MB-157,MCF-7,SKBR-3.Using lentiviral vector alone as negative control,blank group,overexpressing CD82 as CD82-WT group,the mutants at different palmitylation sites were labeled 1,2,3,1+2,1+3,2+3,1+2+3,respectively,with palmitoylation sites of 1,2,3,1+2,1+3,2+3,and 1+2+3,respectively.The expression of CD82 at RNA level was detected by qPCR method.The expression of CD82 at protein level was detected by,Western Blot method.The ability of cell movement and migration was detected by cell scratch test and Transwell test,and CCK8 prescription was used to detect the expression of CD82 at the protein level.Cell cycle was analyzed by flow cytometry.Finally,the expression of related pathway active proteins in CD82 cells was detected by Western Blot method after palmitoylation mutation,and the cell cycle was analyzed by flow cytometry（FCM）,and the cell cycle was analyzed by flow cytometry.To investigate the effects of intracellular signal transduction pathway related to migration and apoptosis of tumor cells.Results:1.The expression of CD82 in three different breast cancer cell lines,MDA-MB-157,MCF-7 and SKBR-3,was detected by Western Blot and qPCR.The results showed that compared with wild type CD82,the expression of CD82 was significantly increased after Cys 5 Cys 74 palmitylation co mutation or Cys 83 site palm acylation mutation.2.The effects of CD 82 palmitoyl mutation on the mobility and migration ability of MDA-MB-157,MCF-7 and SKBR-3 breast cancer cells were detected by scratch and Transwell methods.The results showed that in three different breast cancer cell lines,MDA-MB-157,MCF-7 and SKBR-3,the palmitylation mutation of CD82 at different sites could inhibit the migration of breast cancer cells to different degrees.3.CCK8 was used to detect the effect of CD 82 palmitoyl mutation on the survival rate of MDA-MB-157,MCF-7 and SKBR-3 breast cancer cells.The results showed that the survival rate of SKBR-3 breast cancer cell line was significantly lower than that of wild type CD82 at different sites of CD82 palmitylation mutation.4.Flow cytometry was used to detect the effect of CD 82 palmitoyl mutation on the cell cycle of MDA-MB-157,MCF-7 and SKBR-3 breast cancer cells.The results showed that the palmitylation mutation of CD82 at cys 5 site could block the mitotic G2/M phase in MDA-MD-157 breast cancer cell line compared with CD82-WT.In SKBR-3breast cancer cell line,the palmitylation mutation of CD82 at cys 83 site could block the cell in mitotic G2/M phase.5.Western Blot method was used to study the effect of palmitoyl mutation of CD 82 on the activity of major intracellular signal transduction pathways related to migration and apoptosis of breast cancer cells.The results showed that CD82 palmitoyl mutation had no significant effect on PI3K/AKT signal transduction pathway,and the different palmitoyl site mutations of CD82 could block cell cycle through different apoptosis pathways,thus inhibit migration and decrease survival rate.Conclusion:1.The co-mutation of cys-5 and cys-74 of CD82 or the single point of palmitylation mutation of cys-83 can activate the apoptosis pathway of membrane receptor.2.The cys 5 palmitylation mutation of CD82 could activate the mitochondrial apoptosis pathway.3.The co-mutation of cys 74 and cys 83 of CD82 could activate endoplasmic reticulum apoptosis pathway.