| Objective:To investigate the effect of CAF regulating cell migration and the potential molecular mechanism of human breast cancer.Methods:Bioinformatics methods were used to analyze our previous mRNA and protein microarray data of MCF-10/Twist and MCF-10A/Vector cells, the integrins family were screened out and identified. The expression of ITGB3 in normal, ductal carcinomas in situ(DCIS), invasive and metastasis breast tissue were detected by qRT-PCR. The endogenous high express ITGB3 cell lines of human breast cancer were screened out using qRT-PCR and western blot. ITGB3 stably knockdown cell models of BT549 and Hs578 T were established using lentivirus containing ITGB3-shRNA, and the expression of ITGB3 were validated by q RT-PCR and western blot. The migration ability of ITGB3 knockdown cell models were detected by transwell assay. The ITGB3 knockdown cell models were used to co-culture with NF and CAF, transwell assay and scratch wound healing assay were used to detect the migration ability. ITGB3 were overexpressed in MCF-7 and SK-BR-3 cells, and the expression of ITGB3 were validated by western blot. The ITGB3 overexpression cell models were used to co-culture with NF and CAF, and the migration ability were detected by transwell assay. The mRNA microarray data of NF and CAF were analyzed to screen out the differential expression cytokines, and the outcome were validated using qRT-PCR and ELISA. The association between prognosis of breast cancer patients and CCL18 expression were analyzed using Kaplan–Meier survival analysis. CCL18 expression in NF and CAF of clinic breast cancer samples were detected by qRT-PCR. The interaction between ITGB3 and CCL18 was detected by CO-IP. The expression of CCL18 in CAF was knockdown using CCL18-siRNA, then validated using qRT-PCR, western blot and ELISA. ITGB3 knockdown cell models were co-cultured with siCCL18-CAF, transwell assay was used to detect the migration ability. CCL18 neutralizing antibody was added into CAF condition medium(FBS free),then co-cultured with ITGB3 knockdown cell models, the migration ability were detected by transwell assay. Cytokine CCL18 was directly added into ITGB3 knockdown cell models(FBS free), the migration ability were detected using transwell assay. The downstream signal pathway involved in CCL18-ITGB3 axis and migration related marker E-cadherin, Vimentin were detected using western blot. CCL18 was added into BT549 cells and blocked p38/MAPK signal pathway, the migration ability and E-cadherin, Vimentin were detected using transwell assay and western blot respectively.Results : Integrins family were aberrantly expressed in MCF-10A/Twsit cells compared with MCF-10A/Vector cells. ITGB3 expression was increasing accompany with the malignancy grade of breast cancer development. BT549 and Hs578 T cells endogenous high express ITGB3, whereas MCF-7 and SK-BR-3 did the opposite. The ITGB3 knockdown cell models of BT549, Hs578 T and ITGB3 overexpression cell models of MCF-7, SK-BR-3 were established successfully. CAF could promote ITGB3 positive expression cells migration when they were co-cultured, however, NF could not do the same. CAF could secrete cytokine CCL18, which was aberrantly high. The expression of CCL18 in human breast cancer positive correlated with poor prognosis. The expression of CCL18 in CAF were higher than in NF of clinical breast cancer samples. CCL18 could interact with ITGB3 directly. The ability of CAF promoting ITGB3 positive expression cells migration were down-regulated with the use of CCL18-siRNA or CCL18 neutralizing antibody. CCL18 was directly added into ITGB3 positive cells and the migration ability were up-regulated. CCL18 interacted with ITGB3 and then activated downstream p38/MAPK signal pathway, meanwhile, inhibited E-cadherin expression and promoted Vimentin expression.Conclusions: CAF could secrete cytokine CCL18, that could interact with ITGB3 and activate p38/MAPK signal pathway, then promote cell migration. |
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