Roles Of Embigin In Breast Tumorigenesis And Its Regulation Mechanism In Breast Cancer Cells

Background and objectiveWith the continuous increase in incidence and mortality, cancer has become a major health problem and a leading cause of death among Chinese. Breast cancer is a malignant tumor which has the highest incidence in women cancers. There are 1.8 million new cases of breast cancer and 40 million people died for cancer each year in the world. According to statistics,4.29 million person were diagnosed as cancers in 2015 in China, including 270,000 breast cancer patients.In recent years, the incidence of breast cancer is growing at an annual growth rate of 2.2% and poses serious threat to women’s health.Immunoglobulin superfamily (IgSF) includes a large number of cell adhesion molecules and plays important roles in cell motility, proliferation, differentiation, cell-cell and cell-matrix adhesion, etc. Embigin is a transmembrane glycoprotein belonging to the immunoglobulin superfamily and involves in mouse embryonic development and organ development. Embigin is composed of two extracellular Ig domains, a transmembrane domain (29 amino acids), and a short (47 amino acids) cytoplasmic domain. Recent studies have found that embigin abnormally expressed in various cancer cells, and it may play an important role in the development and progression of cancers.HOXC8 belongs to Homeobox (HOX) protein family and functions as a transcription factor to regulate a series of cell processes including embryonic development, cell adhesion, apoptosis, metastasis and tumorigenesis. Compared to normal tissues, the expression of HOXC8 is higher in cervical cancer, prostate cancer and breast cancer cells, etc, and plays an important role in these cancers development. In our previous study, we found that HOXC8 promotes breast tumorigenesis and metastasis. It also reported that HOXC8 can regulate the expression of embigin in mouse embryonic fibroblasts, but the molecular mechanism still remains unclear. Therefore, this study aims to illustrate the molecular mechanism that HOXC8 regulates embigin expression and the roles of embigin played in proliferation and metastasis of breast cancer cells.Content and methods1. Investigation of the molecular mechanism of HOXC8 in regulation of embigin expression(1) Breast cancer cell lines were lentivirally transduced with HOXC8 shRNA vectors or HOXC8 expression vectorss, and were screened by puromycine to obtain stable HOXC8 overexpression and knockdown breast cancer cell lines. Total RNA were subjected to qRT-PCR and cell lysates were subjected to western blot to detect the levels of embigin mRNA and protein.(2) Embigin promoter luciferase reporter vector was constructed and transfected to HOXC8 overexpression or HOC8 knockdown breast cancer cells. Embigin promoter luciferase activities of these cells were detected to investigate whether HOXC8 regulates embigin expression at its transcriptional level.(3) Breast cancer cell lines were lentivirally transduced with HOXC8 shRNA vectors or HOXC8 expression vectors, and then were treated with 2μg/ml actinomycin. Total RNA was isolated at varying times and then subjected to qRT-PCR to measure the level of EMB mRNA.(4) By analyzing the embigin promoter, we found 12 possible HOX protein binding sites. Based on these possible HOX protein binding sites, we designed four pairs of primers. Chromatin immunoprecipitation experiments were performed in breast cancer cells to determine the binding site of HOXC8 on the promoter of embigin.2. Investigation of the roles of embigin in the proliferation, migration and colony formation of breast cancer cells Breast cancer cell lines were lentivirally transduced with embigin shRNA vectors or embigin expression vectors and were screened by puromycine to obtain stable embigin overexpression and knockdown breast cancer cell lines.(1)MTT assays were performed in these cells to detect the effects of embigin on proliferation of breast cancer cells;(2)Soft agar assays were performed in these cells to detect the influence of embigin on anchorage-independent cell growth of breast cancer cells;(3)Transwell migration assays were performed in these cells to detect the effects of embigin on migration of breast cancer cells.3. Investigation the mechanism of HOXC8 in regulating breast cancer proliferation, migration and colony formation. HOXC8 knockdwn breast cancer cell lines were lentivirally transduced with embigin shRNA vector to establish HOXC8-embigin double knockdown breast cancer cell lines. Cells were screened by puromycine to obtain stable HOXC8-embigin double knockdown cell lines.(1)MTT assay were performed in these cells to detect the influence of HOXC8 knockdown and HOXC8-embigin double knockdown on proliferation of breast cancer cells.(2) Soft agar assay were performed in these cells to detect the influence of HOXC8 knockdown and HOXC8-embigin double knockdown on anchorage-independent cell growth of breast cancer cells.(3) Transwell migration assay were performed in these cells to detect the influence of HOXC8 knockdown and HOXC8-embigin double knockdown on migration of breast cancer cells.Results1. Investigation of the molecular mechanism of HOXC8 in regulation of embigin expression(1) Overexpression of HOXC8 decreased both embigin mRNA and protein levels in breast cancer cells, while HOXC8 knockdown increased both embigin mRNA and protein levels in breast cancer cell.(2) In breast cancer cells transfected with EMB promoter luciferase reporter vectors, HOXC8 overexpression decreased luciferase activities of breast cancer cells, while HOXC8 knockdown increased luciferase activities of breast cancer cells.(3)HOXC8 ecto-expression or shRNA knockdown did not affect embigin mRNA stability.(4) Chromatin Immunoprecipitation was performed with anti-HOXC8 antibody and precipitated DNA was subjected to PCR to examine HOXC8 binding sites in EMB promoter. Data from both Real-time PCR and PCR experiments showed that HOXC8 binds to embigin promoter at the region from nucleotide -2303 to -2315.2. Investigation of the roles of embigin in breast cancer proliferation, migration and colony formation(1) Overexpression of embigin significantly decreased proliferation of breast cancer cells, whereas embigin knockdown enhanced proliferation of breast cancer cells;(2) Embigin overexpression decreased the number of colonies of breast cancer cells distinctly, and embigin knockdown significantly increased the the number of colonies of breast cancer cells;(3) Overexpression of embigin reduced the migration of breast cancer cells significantly, whereas knockdown embigin significantly increased the migration of breast cancer cells.3. Investigation the mechanism of HOXC8 in regulating breast cancer proliferation, migration and colony formation.(1) HOXC8 knockdown significantly decreased the proliferative capacity of breast cancer cells, whereas embigin knockdown can recover the influence of HOXC8 on proliferation capacity of breast cancer cells;(2) HOXC8 knockdown reduced the number of colonies of breast cancer cells, while knockdown embigin can recover the impact of HOXC8 on breast cancer cells;(3) HOXC8 knockdown significantly reduced the migration of breast cancer cells, and this influence can be recovered by embigin knockdown.Conclusion1. HOXC8 binds to the promoter of EMB and functions as a transcription factor to inhibit embigin transcription in breast cancer cells.2. Embigin expression inhibits proliferation, migration and colony formation of breast cancer cells.3. HOXC8 promotes breast tumorigenesis by, at least partially, suppressing EMB transcription in breast cancer cells.