The Role And Its Mechanism Of Mitochondrial Fusion In The Regulation Of Glucose Metabolism Reprogramming In Hepatocellular Carcinoma During Nutrient Deficiency

Mitochondria is an important organelles in the cell metabolism regulation, which continually undergo fusion and fission in response to external environment stimulates. Mitochondria fusion and fission are essential in mammals and their disturbances are associated with several diseases, such as neurodegenerative diseases, cardiovascular disease, and metabolic disease. Recently, studies have showed that tumor cells always have imbalanced mitochondrial fusion and fission, which is involved in tumor progression. Hepatocellular carcinoma is a kind of solid tumor always with reduced nutrients supply. Tumor cells select effective ways to adapt to the nutrition-limited conditions by energy metabolism reprogramming. Our recent work showed that hepatocellular carcinoma cells have increased mitochondial fusion under nutrition-limited conditions. The role of mitochondrial fusion in energy metabolism regulation of HCC cells under nutrition-limited conditions is currently unclear. Aims:1. To explore the mechanism of mitochondria fusion induced by nutrient deficiency in hepatocellular carcinoma. 2. To explore the role of mitochondria fusion in the regulation of energy metabolism in hepatocellular carcinoma cells. 3. To study the biological significance of mitochondria fusion-mediated energy metabolism reprogramming under nutrition-limited conditions in hepatocellular carcinoma. Methods:1.Mitochondria morphology was assessed by Mito-tracker Green Staining and Transmission Electron Microscope(TEM) to confirm the regulation of mitochondria fusion induced by nutrient deficiency in HCC tumor cell lines and tumor tissues. 2.The expression of major molecules in the regulation of mitochondrial fusion and fission were measured by Quantitative real-time polymerase chain reaction(qRT-PCR) and Western Blot to study the molecular mechanisms involved in mitochondria fusion under nutrition-limited conditions. 3.The role of mitochondria fusion in regulating glycolysis and oxidative phosphorylation were assessed by qRT-PCR, Western Blot and gas chromatograph-mass spectrometer(GC-MS) analysis under nutrition-limited conditions. 4. TEM, Blue native-PAGE, Co-IP, qRT-PCR and Western Blot were used to determine the molecular mechanisms involved in promotion of Oxidative Phosphorylation and inhibition of glycolysis by mitochondrial fusion in hepatocellular carcinoma cells under nutrition-limited conditions. 5.The role of mitochondria fusion in the promotion of tumor cell survival were assessed by flow cytometry(FCM) and clony formation assay under Nutrition-Limited Conditions. 6. The role of mitochondria fusion in tumor growth was assessed by Xenograft tumor assay under Nutrition-Limited Conditions in vivo. 7.The expression of p-DRP1S937 in HCC tissues was detected by IHC analysis and the relationships between p-DRP1S937 expression and the clinical pathological parameters and prognosis were analyzed. Results:1.Mito-tracker Green Staining showed elongated mitochondria under Nutrition-Limited Conditions; Transmission electron microscope(TEM) images showed that tumor tissues from central zone have elongated mitochondria than those from peritumoral zone. 2.No significant change was found in the expression of major molecules mediating mitochondrial fusion and fission in HCC cells before and after nutrient deprivation, while mitochondria-located DRP1 was reduced due to its phosphorylation by PKA at Ser637, which result in elongated mitochondria. 3.Nutrition deficiency-induced mitochondrial elongation induced a metabolic shift from glycolysis to Oxidative Phosphorylation in HCC cells. 4.Nutrition deficiency-induced mitochondrial elongation promotes Oxidative Phosphorylation through tighting the cristae junction and assembly of respiratory complexes, while inhibits glycolysis through activation of NAD+/SIRT1/HIF-1α signaling by Oxidative Phosphorylation in HCC cells. 5.Nutrition deficiency-induced mitochondrial elongation inhibits apoptosis and promotes colony formation of in HCC cell. 6. Nutrition deficiency-induced mitochondrial fusion promotes tumor growth of HCC in vivo. 7.The expression of p-DRP1S637 is positively correlated with the differentiation, TNM stage and serum AFP levels of HCC patients, and predicts poor survival outcomes of HCC patients. Conclusions:1.Nutrition deficiency induces mitochondria elongation in HCC cells. 2. Phosphorylation of DRP1 by PKA at Ser637 mediated nutrition deficiency-induced mitochondria elongation in HCC cells. 3. Mitochondrial elongation promotes HCC cell survival in nutrition-limited conditions by regulating energy metabolism through facilitating the cristae remodeling. 4. p-DRP1S637 is associated with poor prognosis of HCC patients and may be a potential biomarker for HCC prognosis.