The Effect And Mechanism Of FAM64A On The Progress Of Epithelial-Mesenchymal Transition Of Breast Cancer

There was no evidence of the effect of Family with Sequence Similarity 64,Member A (FAM64A) on tumor. We found the upregulating of FAM64A in kinds of tumor tissues by searching The Cancer Genome Atlas (TCGA) data base. We also found the relation between FAM64A and poor prognosis through analysis on Gene expression-based Outcome for breast cancer Online (GOBO). As the main cause of the death of breast cancer patients was metastasis, it was conducted that, the relationship between FAM64A and metastasis might exist.Epithelial-mesenchymal transition (EMT) is a cell process of losing epithelial phenotype and acquiring mesenchymal phenotype. The characteristics of EMT includes the loss of cell polarity and cell-cell contact (epithelial phenotype), the enhancement of cell invasion and migration (mesenchymal phenotype). EMT was found both in the embryonic development and pathological process. During the process of metastasis, EMT was believed to be an important step when the tumor cells detach from primary position and implant in the distant tissue. The activation of TGF-β signaling pathway was considered play an important role in the process of EMT. Our previous studies demonstrated that, the expression of FAM64A could be upregulated by adding of TGF-β in vitro. It might suggest the effect of FAM64A on TGF-β-depended EMT.In this study, we found the relationship between FAM64A and poor prognosis of breast cancer through online database. IHC assays and qRT-PCR were used to evaluate the relationship between the expression of FAM64A and breast cancer, and we found the over-expression of FAM64A in breast cancer tissues. In addition, in vitro experiments were performed to investigate the effect of FAM64A on breast cancer cells. The expression of FAM64A were controlled by si-RNA and transfection techenique in vitro, we found FAM64A played a necessary role in the cell morphology and the process of invasion and migration. The qRT-PCR and Western Blotting were performed to investigate the effect of FAM64A on the expression of key proteins during the process of EMT.To further explore the molecular mechanism of FAM64A promoting cell EMT,we studied the relationship between FAM64A and E-cadherin. It was found that FAM64A inhibited the activation of CDH1 gene (E-cadherin coding gene) promoter by luciferase assay and RT-PCR, thus inhibiting the transcription of CDH1 gene.Further studies by CHIP showed that FAM64A could bind to the CDH1 gene.Through the co-IP test, it was found in the exogenous and endogenous system, that FAM64A can be directly combined with Twistl. After interference with Twistl expression, FAM64A could not bind to CDH1 gene, and its inhibition of CDH1 promoter activation and expression would also be reduction. Since Twistl can bind to the promoter region of E-cadherin by the NuRD complex, it was demonstrated that FAM64A can be combined with Twistl to form a complex, which could bind with E-cadherin promoter to inhibite the expression of E-cadherin, and thus promote the process of cell EMT.