Study On Energy Metabolism During EMT Transformation Induced By TGF-?1 In Breast Cancer Cells

Objectives: To enhance the transformation of breast cancer cells MCF-7 from epithelial cells to mesenchymal cells by adding TGF-?1 Through the establishment of a stable EMT cell model,to explore the MCF-7 EMT transformation process of energy metabolism changes and regulatory mechanisms.Methods: MCF,7 cells were cultured in medium containing TGF-?1 (2 ng / ml) for 9 days, followed by normal medium for 9 days. In this process, MCF-7 cells complete the process of transformation from epithelial cells to interstitial cells and subsequent transformation from stromal cells into epithelial cells. In the experiment, we used qPCR and western to detect the expression of E-cadherin, stromal cell marker protein(Vimentin) and transcription factor Snail 1 in EMT epithelial cells, and detected by cell invasion assay; The expression of PDH, LDH, NDUFB8, TFAM and COX1 were detected by western blot; The number of mitochondria and mitochondrial DNA copy number were detected by flow cytometry and qPCR; The expressions of FASN, CPT1 and CD36 were correlated with the expression of lipid metabolism related genes and proteins; The expression of related signal pathway proteins, which may cause lipid metabolism changes,was detected. Finally,20 cases of breast cancer clinical samples were collected and analyzed by immunohistochemistry. The changes of expression of lipid metabolism related proteins in breast cancer tissues were examined. Through the above research, we get the following results:Results(1) TGF-?1 induced the transformation of MCF-7 from epithelial cells to interstitial cells. During the transformation, the changes of cell morphology and adhesion were observed, and the expression of E-cadherin was decreased, and Vimentin and Snaill are increasing expression levels.(2) TGF-?1 induce MCF-7 EMT is decreasing the cell proliferation ability, but the ATP level increased and the cell invasion ability increased.(3) The expression of NDUFB8, TFAM and COX1 in MCF-7 was significantly increased in the process of EMT, and the number of mitochondria and mitochondrial DNA were also increased. These results indicated that the activity of mitochondrial oxidative phosphorylation was enhanced during EMT transformation.(4) In the process of EMT transformation, the expression of FASN was decreased, the expression of CPT-1 and CD36 increased, the synthesis pathway of cell fatty acid decreased and fatty acid p-oxidation increased.(5) The expression of P-AMPK was decreased and P-AMPK could increase the expression of CPT-1 and increase the intracellular fatty acid oxidation by inhibiting the expression of ACC.(6) Immunohistochemical analysis of 20 cases of breast cancer clinical samples showed that high expression of FASN in E-cadherin overexpressing epithelial cells in breast cancer tissues and high expression of CPT-la in Vimentin overexpressing stromal cells In the breast cancer tissue epithelial cells and stromal cells in the presence of significant partition in the epithelial cells in the synthesis of fatty acids to enhance fat storage, and interstitial cells to strengthen the fatty acid ?-oxidation, resulting in a large number of ATP for cell invasion and metastasis. These results suggested that different areas of tumor tissue and different cell types have different metabolic pathway.Conclusion: TGF-?1 induced MCF-7 in the process of EMT, the cell proliferation was slowed down, the cell invasion ability increased, the ATP level increased, the mitochondrial oxidative phosphorylation activity increased, the fatty acid ? oxidation increased, and the fatty acid synthesis decreased. At the same time breast cancer tissue also detected in the epithelial cell fatty acid synthesis to strengthen, conducive to fat storage, and interstitial cells in the fatty acid P oxidation enhanced, resulting in a large number of ATP in favor of cell invasion and metastasis. Different areas of tumor tissue and different cell types of metabolic pathway are different. This study could help us to understand of the metabolic characteristics of tumor metastasis, lay the foundation for the study of the relationship between energy metabolism and metastasis during EMT transformation.