| Breast cancer is the most common malignant tumor for women, which has been ranked first for women’s death of tumor. Although the incidence of breast cancer in China is lower than that in western countries, in recent years, with the improvement of people’s living standards and the changes of lifestyle, the nation-wide average of breast cancer mortality has reached to26.1per million. Nowsdays the therapeutic options for breast cancer mainly include surgery, radiotherapy, chemotherapy and endocrine therapy. Clinical data indicate, despite comprehensive surgery treatment or chemotherapy, there are still60-70%of Ⅱ-Ⅲ stage patients relapsed in five years, and distant metastases in patients is proven to be the leading cause of death.Recent studies indicate that the epithelial-mesenchymal transition (EMT) of breast cancer cells is the main reason for its metastasis. EMT is the process that the polarity cells gain the ability to move freely in cell matrix and characterized by shifting from epithelial phenotype to mesenchymal phenotype. Breast cancer cells get stronger ability of invasion and metastasis in the EMT process, then break through extracellular matrix and basement membrane of blood vessels and form the secondary distant metastases through the circulatory system. Therefore, the molecules involved in regulating EMT in breast cancer cells can be used as the molecular markers of breast cancer prognosis. In-depth study of the basic molecular mechanisms of invasion and metastasis of breast cancer cells would lead to appropriate individualized treatment, which is the current hot and difficult field of breast cancer research.Ubiquitin is a conserved polypeptide consisted of76amino acid residues and its function in the protein degradation pathway is strictly regulated by time and space. With the supply of ATP, ubiquitin is activated by ubiquitin-activating enzyme (E1), and transferred to the ubiquitin-conjugating enzyme (E2), then connected to specific substrate by ubiquitin ligase (E3). Finally, the ubiquitinated substrate gets into proteasome for degradation. In this process, E3recruits specific substrate and mediates the transport of ubiquitin to the target protein from E2, therefore E3is regarded as a key player in protein degradation and determines the specificity of the reaction. CUL4A is a kind of ubiquitin E3ligase, which combines with DDB1and Rocl to form the E3complex (CUL4A-DDB1-Rocl). Our understanding of CUL4A originated from studies in breast tumors, and CUL4A gene amplification or overexpression was found in primary breast cancer and subsequently in ovarian cancer, lung cancer, squamous cell carcinoma, adrenal cortical carcinoma, medulloblastoma, hepatocellular carcinoma, and malignant pleural mesothelioma. Moreover, CUL4A overexpression was proven to promote cancer cell proliferation and has a close association with the lower overall survival rate and disease-free survival rate, suggesting that abnormal regulation of CUL4A may promote the tumor formation. However, Whether CUL4A is able to regulate EMT and invasion of breast cancer cell has not been reported. The main purpose of this study was to clarify the role of CUL4A in regulating the EMT, invasion and metastasis in breast cancer cells, and possible molecular mechanisms. The successful implementation of this study not only enriched the CUL4A biological function in breast cancer, but also shed linght on exploring the prevention and treatment of the invasion and metastasis of breast cancer.Part I. The correlation of E3ubiquitin ligase CUL4A and tumor metastasis[Object]To study the expression of CUL4A in various malignant cells and different tumor types, we used some commonly-used cell lines and tissue microarray. Moreover, analysis of the relationship between CUL4A and tumor metastasis may lay the foundation to further define the role of CUL4A in breast cancer invasion and metastasis.[Methods]1. Detect the expression of CUL4A by qRT-PCR and Western blot in commonly-used breast cancer cell lines preserved in the laboratory.2. Detect the expression of CUL4A in normal breast tissue, carcinoma in situ and metastatic carcinoma by immunohistochemistry with Biomax Bio’s breast tissue microarray (BR954).3. Detect the expression of CUL4A in normal gastric tissue, carcinoma in situ and metastatic carcinoma by immunohistochemistry with Biomax Bio’s gastric tissue microarray (ST8041). 4. Detect the expression of CUL4A in normal ovarian tissue, carcinoma in situ and metastatic carcinoma by immunohistochemistry with Biomax Bio’s ovarian tissue chip (OV2089).5. Detect the expression of CUL4A in normal colon tissue, carcinoma in situ and metastatic carcinoma by immunohistochemistry with Biomax Bio’s colon cancer tissue microarray (C020814).[Result]1.qRT-PCR results showed that mRNA expression level is low in normal CUL4A breast epithelial cells (MCF10A), while higher in the non-invasive breast cancer cells (MDA-MB-468, MCF7, HCC1569), and highest in the invasion of breast cancer cells (MDA-MB-231.BT549).2. Western blot analysis showed protein expression levels of CUL4A in various types of cell lines are highly consistent with that of mRNA expression.3. The detection of CUL4A protein expression level in breast cancer tissue array by immunohistochemistry showed that the expression level of CUL4A is low in normal breast tissue, high in carcinoma in situ, and highest in breast cancer with distant metastases.4. The detection of CUL4A protein expression level in gastric cancer tissue array by immunohistochemistry showed that the expression level of CUL4A is low in normal gastric tissue, high in carcinoma in situ, and highest in gastric cancer with distant metastases.5. The detection of CUL4A protein expression level in ovarian cancer tissue array by immunohistochemistry showed that the expression level of CUL4A is low in normal ovarian tissue, high in carcinoma in situ, and highest in ovarian cancer with distant metastases.6. The detection of CUL4A protein expression level in colon cancer tissue array by immunohistochemistry showed that the expression level of CUL4A is low in normal colon tissue, high in carcinoma in situ, and highest in colon cancer with distant metastases.[Conclusion]1. CUL4A expression levels are different in various breast cell lines:the mRNA and protein levels in non-invasive cell lines are much lower than that in invasive cell lines, indicating that CUL4A is closely related to the invasive property of breast cancer cells.2. Tissue array immunohistochemistry results prove that in different types of tumors, CUL4A expression increased significantly in tumors with distant metastasis comparing with in situ carsinomas, indicating that CUL4A is closely associated to cancer metastasis.Part II. Role of CUL4A in promoting epithelial-mesenchymal transition and metastasis of breast cancer cell[Object]The data in the first section indicate that CUL4A may promote invasion and metastasis of breast cancer cells. In this section, we tested whether CUL4A have the ability of inducing EMT and metastasis-of breast cancer cells by in vitro experiments and nude mice metastasis model experiment.[Methods]1. CUL4A over-expressing and control cell lines (MDA-MB-468-pBabe, MDA-MB-468-pBabe-CUL4A, MCF7-pBabe, MCF7-pBabe-CUL4A) were constructed by transfecting with pBabe-CUL4A and the control plasmid with retro virus. After the puromycin screening, the changes of CUL4A expression were verified by qRT-PCR and Western blot.2. CUL4A silence and control cell lines (MDA-MB-231-pSuper, MDA-MB-231-pSuper-shCUL4A, BT549-pSuper, BT549-pSuper-shCUL4A) were constructed by transfecting with pSuper-shCUL4A and the control plasmid by retrovirus. After the puromycin screening, the changes of CUL4A expression were verified by qRT-PCR and Western blot.3. To observe the role of CUL4A in regulating EMT process in breast cancer cells, the cell morphology were observed through the light microscope and the expression of some related markers (i.e. E-cadherin, a-catenin, N-cadherin, Vimentin) were detected with the application of qRT-PCR, Western blot, confocal immunofluorescence in all above CUL4A stable silencing and over-expressing cell lines.4. With the stable transfected cell lines, we tested the role of CUL4A in regulating the migration ability of breast cancer cells by cell wound scratch assay and transwell chamber migration assay and the invasion ability by Matrigel invasion assay. 5. To study whether the up-regulated CUL4A can promote the invasion and metastasis ability of breast cancer cells, we established xenograft models and distant metastasis model of nude mouse with the application of the constructed over-expressing and control cell lines and observed the metastasis of tumors.6. To study whether the down-regulated CUL4A can inhibit the invasion and metastasis ability of breast cancer cells, we established xenograft models and distant metastasis model of nude mouse with the application of CUL4A silencing and control cell lines and observed the metastasis of tumors.7. To further verify the changes of EMT, which has been proven in vitro, we detected the expression level of CUL4A, E-cadherin and Vimentin in tumors and metastases by qRT-PCR, Western blot and immunohistochemical staining methods.[Result]1. The CUL4A stable overexpression and control breast cancer cell lines were successfully established (MDA-MB-468-pBabe, MDA-MB-468-pBabe-CUL4A, MCF7-pBabe, MCF7-pBabe-CUL4A).2. The CUL4A silencing and control breast cancer cell lines were successfully established (MD A-MB-231-pSuper, MDA-MB-231-pSuper-shCUL4A, BT549-pSuper,BT549-pSuper-shCUL4A).3. CUL4A promoted the expression of mesenchymal cell markers such as N-cadherin, Vimentin etc., and inhibited the expression of markers of epithelial cell such as E-cadherin, a-catenin etc..4. CUL4A promoted the morphological transformation of breast cancer cells from epithelial to mesenchymal type.5. CUL4A enhanced the mobile and invasive capacities of breast cancer cells in vitro.6. In vivo metastases experiments showed that CUL4A promoted the motility, invasion and metastasis ability of breast cancer cells.[Conclusion]1. CUL4A can regulate the EMT process in breast cancer cells.2. CUL4A promotes migration and invasion of breast cancer cells in vitro.2. CUL4A can promote metastasis of breast cancer cells in vivo.Part III. The molecular mechanism of CUL4A in promoting EMT, invasion and metastasis of breast cancer cells[Object]Clinical specimen analysis and experimental data showed CUL4A has the%bility to promote EMT, invasion and metastasis of breast cancer cells, but its specific mechanism has not been reported. The major object of this part was to clarify its possible molecular mechanisms.[Methods]1. Detect the effect of CUL4A on MDA-MB-468gene expression profile with Affymetrix GeneChip Human U133Plus2.0cartridge.2. Verify the expression of ZEB1which has been proven to be close related with EMT by gene chip in more stable transfected breast cancer cell lines by qRT-PCR and Western blot.3. Screen H3methylation status by Western blot.4. Detect the effect of CUL4A on the binding of H3K4me3to ZEB1promoter by chromatin immunoprecipitation and qPCR.5. Stably ZEB1silencing cell line and control cell line were constructed in MDA-MB-468-pBabe-CUL4A cells by retroviruses transfection system.6. Detect the expression changes of EMT markers in the above-mentioned cell lines by Western blot.7. To study whether the down-regulated ZEB1can inhibit the CUL4A induced migration and invasion of breast cancer cells in vitro, we tested the migration ability by cell wound scratch assay and Transwell chamber migration assay and the invasion ability by Matrigel invasion assay in above cell lines.8. To study whether the down-regulated ZEB1can inhibit the CUL4A induced metastasis of breast cancer cells in vivo, we established xenograft distant metastasis model of nude mouse with the application of the constructed ZEB1silencing and control cell lines and observed the tumor metastasis.9. Detect the expression of ZEB1in normal breast tissue, carcinoma in situ and metastatic carcinoma by immunohistochemistry with Biomax Bio’s breast tissue microarray (BR954). Correlation of ZEB1and CUL4A was analyzed.[Result]1. Gene chip result showed that ectopic CUL4A expression could promote ZEB1expression which is a key regulator in EMT. Results from qRT-PCR and Western blot in the stable transfected cell lines are consistent with gene chip.2. Detection of H3methylation in the stable transfected cell lines proved that CUL4A could promote H3K4trimethylation.3. Chromatin immunoprecipitation proved that CUL4A could promote the binding of H3K4me3to ZEB1promoter.4. Interference of ZEB1expression could inhibit CUL4A overexpression-induced EMT.5. Interference of ZEB1expression could inhibit CUL4A induced changes in cell mobility and invasion.6. In vivo experiments proved that interference of ZEB1expression could inhibit CUL4A induced metastasis of breast cancer cells.7. Tissue chip analysis of ZEB1shows that ZEB1expression is high in metastatic breast cancer, and lower in normal breast tissue and carcinoma in situ.[Conclusion]1. CUL4A promotes EMT by promoting the expression of ZEB1.2. CUL4A promotes transcriptional expression of ZEB1by modification of H3K4me3... |
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