关键词: 胆碱激酶α, 胶质母细胞瘤, 线粒体功能, Mdivi-1, 活性氧, 线粒体膜电位, 增殖, 凋亡

Abstract: BACKGROUND: Choline kinase alpha is a key enzyme in phospholipid metabolism, involved in the synthesis of phosphatidylcholine, and plays an important role in maintaining cell membrane integrity and signal transduction. Research has shown that choline kinase alpha is highly expressed in various tumors and is closely related to cell proliferation, metabolic reprogramming, and tumor progression. As a potential therapeutic target, the role of choline kinase alpha in tumor metabolism and mitochondrial function still needs further exploration. 
OBJECTIVE: To evaluate the effects and the underlying mechanisms of choline kinase alpha on the proliferation and apoptosis of glioma U87MG and U251 cells. 
METHODS: Short hairpin RNA of choline kinase alpha and its empty vector control were transfected into U87MG and U251 glioma cells. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial structure and functional protein levels were assessed by western blot assay. Reactive oxygen species levels in cells were measured using a reactive oxygen species fluorescent probe. Mitochondrial membrane potential was assessed with a JC-1 assay. Intracellular adenosine triphosphate levels were measured by chemiluminescence. Cell proliferation was evaluated using a CCK-8 assay. Apoptosis levels were analyzed by flow cytometry. The mitochondrial fission inhibitor Mdivi-1 was used to protect the mitochondrial function of the choline kinase α-silenced lentiviral cells. Finally, U87MG cells were subcutaneously injected to construct a subcutaneous tumor model in nude mice. The tumor growth in nude mice was observed before and after choline kinase alpha silencing and after the use of the mitochondrial fission inhibitor Mdivi-1. 
RESULTS AND CONCLUSION: (1) Compared with the empty control group, the mitochondria of U87MG and U251 cells in the choline kinase alpha silencing lentivirus group exhibited significant structural abnormalities in mitochondria, such as vacuolization and cristae disruption. The expressions of mitochondrial structure and function-related proteins TOM20, ACO2, and ATP5A were significantly decreased (P < 0.01, P < 0.001), the expression of SOD2 was significantly increased (P < 0.01, P < 0.000 1), the fluorescence intensity of reactive oxygen species was significantly increased (P < 0.01), the mitochondrial membrane potential and adenosine triphosphate level were significantly decreased (P < 0.01, P < 0.001), the cell proliferation ability was reduced (P < 0.01), and the apoptosis level was increased (P < 0.001). (2) Following Mdivi-1 treatment, the fluorescence intensity of reactive oxygen species in U87MG and U251 cells decreased (P < 0.05, P < 0.01), mitochondrial membrane potential and adenosine triphosphate levels were significantly restored (P < 0.05, P < 0.01, P < 0.001), cell proliferation ability was improved (P < 0.05, P < 0.01), and apoptosis level was decreased (P < 0.05). (3) In addition, the in vitro subcutaneous tumor formation experiment of nude mice showed that compared with the empty control group, the mass and growth rate of subcutaneous tumors formed by U87MG cells in the choline kinase alpha silencing lentivirus group were significantly reduced (P < 0.000 1). After Mdivi-1 treatment, the mass and growth rate of tumors were significantly increased (P < 0.000 1). (4) The results show that choline kinase alpha silencing affects the proliferation and apoptosis of glioma cells by inducing mitochondrial dysfunction.

Key words: holine kinase alpha, glioblastoma, mitochondrial function, Mdivi-1, reactive oxygen species, mitochondrial membrane potential, proliferation, apoptosis