Primary Study Of Foxal Interacting Proteins And The Regulation Of Transcription Factor FOXA1 In The Process Of Epithelial-Mesenchymal Transition In Breast Cancer Cells

Breast cancer is one of the most common cancers which threatening the female health, and the global incidence of this disease has an upward trend since the 1970s. Since breast cancer cells have three unique features:unlimited growth?transformation and metastasis, it is often very difficult to cure if breast cancer was checked out lately. Therefore, it's important to explore various types of breast cancer cell signaling pathways and find the target gene.Transcription factor FOXA1 is one of forkhead box transcription factor family members, it's related to cell proliferation, differentiation, embryonic growth, organ development, tumor development and some other physiological processes. Numerous studies showed that, FOXA1's expression was highly interconnected with tumor molecular typing and malignancy in cancer cells. So we guess FOXA1 can regulate the expression of genes which related to cell EMT, maybe FOXA1 was one of the key regulatory factors. Our research was mainly about FOXA1 regulation mechanism and interacting proteins in breast cancer cells.We tested the protein expression levels of FOXA1,E-cadherin, vimentin and ?-actin from dozens of clinical samples, we found in clinical samples FOXA1 was positively correlated with the expression of epithelial markers E-cadherin, and it was negatively correlated with the expression of mesenchymal markers Vimentin. Then we detected the protein and gene expression of FOXA1, E-cadherin, vimentin from eight different types of breast cancer cells, We found E-cadherin's expression was high in breast cancer cells which expressing highly FOXA1, whereas the expression of vimentin was low. So we found that the transcription factor FOXA1 was correlated with epithelial phenotype in breast cancer cells.ZEB2 can inhibit the expression of E-cadherin, we hypothesized that FOXA1 may regulate ZEB2's transcription.Depending the expression levels of FOXA1, we used the E-type breast cancer MCF-7 cells and triple-negative breast cancer cells MDA-MB-468 cells as model cells, We liposomal transfected FOXA1's plasmid to increase expression FOXA1 in triple negative breast cancer cell line MB-468, then we found in these cells the expression of ZEB2 and vimentin was decreased, but the epithelial markers E-cadherin's expression was increased. Then reporter gene experiments by luciferase confirmed that FOXA1 can be directly incorporated in the target gene ZEB2's promoter and inhibited its transcription.In order to find FOXA1's interacting proteins and explore its function in signaling pathways in breast cancer cells, we have established the technical assays of His tag pull-down technology combined with mass spectrometry. First, we constructed the eukaryotic expression vector pcDNA3.1-His-FOXA1 and then transfected it in HEK293T cells to increase His-FOXA1 expression level. The FOXA1's protein complex was isolated using His-tag pull-down assay. Then the components of the FOXA1 complex were identified by mass spectrometry. we screened out seven FOXA1's interacting protein by Data analysis. These studies provided a technical foundation for further research of FOXA1's function in signaling networks and regulation in breast cancer cells.