结肠上皮细胞NCM460中线粒体DNA部分缺失诱导其恶性转化及免疫逃逸OA

Objective:To clarify the effects of mitochondrial DNA(mtDNA)partial depletion on colonic epithelial cell NCM460.Methods:Mitochondrial DNA partially depleted cell model(mtDNA-reduced)and reversion model(reverted)were established by ethidium bromide(EB)induction.Mitochondrial function was evaluated by CCK-8(Cell Counting Kit-8)assay,reactive oxygen species(ROS)and mitochondrial membrane potential(JC-1)detection.Metabolic phenotypes were assessed by glucose,lactate and glycolytic key enzyme activity assays.Cell invasion and epithelial-mesenchymal transition(EMT)were detected by Transwell invasion assay and Western blot.Anoikis resistance was examined by polyHEMA coated culture.The PI3K/Akt/mTOR signaling pathway and programmed death ligand 1(PD-L1)expression were detected,and verified by PI3K inhibitor LY294002.Results:GEPIA(Gene Expression Profiling Interactive Analysis)database analysis showed that mtDNA-encoded genes were down-regulated in colorectal cancer(P＜0.05).Tissue microarray showed that cytochrome coxidase subunit 1(COX1)protein was highly expressed in normal colonic tissues but lowly expressed in cancer tissues(P＜0.05).Mitochondrial DNA-reduced NCM460 cells showed impaired mitochondrial function(decreased growth rate,reduced ROS generation,and decreased mitochondrial membrane potential,P＜0.05),exhibited the Warburg effect(increased glucose uptake,enhanced lactate secretion,and increased glycolytic key enzyme activity,P＜0.05),enhanced invasion capacity(P＜0.05)with EMT,and resisted anoikis(P＜0.05).The PI3K/Akt/mTOR pathway was activated and PD-L1 expression was upregulated in these cells.After treatment with LY294002,the glucose content showed a decreasing trend(P＞0.05),the cell activity decreased(P＜0.05),and PD-L1 expression was downregulated(P＜0.05).Conclusion:Partial depletion of mtDNA in colonic epithelial cell NCM460 can induce malignant transformation and immune escape.