The Effects Of CRABP2 On 4-amino-2-trifluoromethyl-phenyl Retinate Induced Cell Differentiation And Inhibit Proliferation And Its Mechanism On Breast Cancer

Breast cancer is a malignant tumor,which seriously threatens women's health.The current treatments for breast cancer all have limitation such as surgical therapy,systemic chemotherapy,radiation therapy and endocrine therapy based on different indications.In recent years,with the concept of induced differentiation in clinical cancer treatment continues to deepen,anti-tumor effects of retinoids have increasingly been the concern of scholars at home and abroad.4-Amino-2-Trifluoromethyl-Phenyl Retinate?ATPR?is a new retinoid derivative designed and synthesized in our team based on the differentiation agent all-trans-retinoic acid?ATRA?,which has an anti-cancer effects on a variety of tumors.Our previous studies have demonstrated that ATPR have an anti-proliferation and pro-differentiation effects on many tumor cell lines,including breast cancer cell lines.In order to explore the feasibility of ATPR in clinical treatment of breast cancer,it is necessary to study the effects and mechanisms of ATPR on differentiation and proliferation of breast cancer cells.It has been reported in the literature that cellular retinoic acid-binding protein-2?CRABP2?plays an important role in the retinoic acid signaling pathway and retinoic acid-induced differentiation.Does it participate in the regulation of ATPR induced breast cancer cell differentiation and inhibition of proliferation?In this study,breast Cancer pathology tissues and breast cancer cell lines were selected as research objects,Effects of CRABP2 on ATPR-induced breast cancer cell proliferation and differentia-tion and its mechanism of action were explored from tissue to cellular level.The expression of CRABP2 protein in breast cancer by immunocytochemistry staining;The expression of CRABP2 protein in breast cancer MCF-7 cells,MDA-MB-231 cells,MDA-MB-435 cells and MDA-MB-453 cells was observed by immunofluorescence.The effect of ATPR on the proliferation of those four breast cancer cells was observed used MTT assay after 24,48 and 72 h,respectively.The CRABP2 stable silent model was established in MCF-7 cells andthen cells were divided into three different group designated:control group,the shRNA-NC group and the CRABP2 silencing group?sh-CRABP2 group?.After treating with ATPR(1×10^-5mol/L)for 5 days,cell morphological changes were observed by using HE staining,oil red O staining were used to observe the morphological changes of cells.MTT assay was used to detect the cell proliferation.The expression of PCNA was detected by immunocytochemistry?ICC?.Flow cytometry?FCM?was used to detect breast cancer cell differentiation maker ICAM-1 expression and cell cycle changes.Transwell method was used to determine cell migration and invasion.Immunoprecipitation?CO-IP?assay was used to detect the interaction relationship between CRABP2 protein and HuR protein.The expression of HuR and ICAM-1 protein,and the phosphorylation of MEK-1,ERK and STAT3 protein were detected by western Blot.The results of this study showed that:The CRABP2 protein is expressed in poorly differentiated,moderately differentiated and well-differentiated breast cancer,and increased with the pathological grade of breast cancer,the least expressed in poorly differentiated breast cancer,moderately differentiated breast cancer is stronger than low Differentiated breast cancer,the most expressed in well-differentiated breast cancer.The results showed that CRABP2 protein expression in these 4 breast cancer cells?MCF-7cells,MDA-MB-453 cells,MDA-MB-435 cells,MDA-MB-231 cells?showed a decreasing trend.ATPR significantly inhibited MCF-7 cells proliferation in a concentration-and time-dependent manner.Interestingly,the inhibitory effects of ATPR on MDA-MB-231,MDA-MB-435 and MDA-MB-453 cells proliferation were relatively weaker than that in MCF-7 cells.Moreover,the results of HE staining,oil red O staining,ICC and FCM also showed that ATPR could significantly induce differentiation and inhibit proliferation,and its effect was related to CRABP2 expression.Silencing CRABP2 expression could decrease ATPR pro-differentiation and inhibited proliferation effects.Migration and invasion assays showed that ATPR could significantly reduce the number of migratory and invasive cells in MCF-7 cells,but have no significant effect after silencing CRABP2 gene.The results of CO-IP showed that there was interaction between CRABP2 and HuR in MCF-7 cells,and this interaction was weakened after ATPR treatment.Western Blot results showed that compared with control group,while the levels of HuR,p-MEK-1 and p-ERK protein were decreased,and the levels of p-STAT3 and ICAM-1 were increased in sh-CRABP2group and ATPR group.In conclusion,our results showed that ATPR has an obviously induced effect on differentiation,and inhibitory effect on proliferation and metastasis in breast cancer cells with high CRABP2 expression.Moreover,ATPR induces breast cancer cells differentiation by activating HuR-MEK-1/ERK-STAT3-ICAM-1 signaling pathway under the mediation of CRABP2 protein.