| Part1 The value of lipid metabolism in predicting prognostic risk in gastric cancer patientsBackground: Alterations in lipids metabolism in cancer cells are increasingly being recognized,but the application of lipid metabolism in the prognosis of gastric cancer(GC)has not yet been explored.Methods: A total of 204 lipid metabolism relative genes were analyzed in a GC cohort from TCGA,while four independent cohorts from GEO were used for external validation and one cohort from Wuhan Union Hospital was applied to immunohistochemical validation.Difference and enrichment analyses were performed between gastric cancer and normal tissue.Further,a robust LASSO-Cox proportional hazard regression model was applied to select prognostic genes and to construct a gastric cancer score-based genes profiling.Results: Ether lipid metabolism,glycerophospholipid metabolism,and glycerolipid metabolism were up-regulated and Fatty acid ? oxidation,oxidation-reduction process,lipid oxidation,oxidoreductase activity,and other process were reduced in GC rather to normal tissue.A genes profiling composed of expression of AGPAT3,AKR1B1,PLD1,and UGT8 suggested 3 groups with a significant difference in overall survival(HR = 2.00,95%CI = 1.54–2.59,p < 0.001).Further,martingale residuals of profiling plots and Schoenfeld individual test plots showed balanced hazard proportionality.The genes profiling was successfully validated in an independent cohort(HR: 1.48,95%CI: 1.11-1.97,p < 0.001).Moreover,genes profiling performed well in the immunohistochemical cohort(HR: 2.16,95%CI: 1.42-3.31,p < 0.001).Conclusion: We explored lipid metabolism is reliable and clinically applicable in predicting the prognosis of gastric cancer based on a novel prognostic genes profiling.Part2 PLD1 regulation of free fatty acid metabolism to counter nutrient stress in gastric cancer cellsBackground: To investigate the clinical significance and specific mechanism of PLD1 regulation of free fatty acid metabolism on nutritional stress in gastric cancer cells.Methods: The association between PLD1 expression and prognosis of gastric cancer patients was retrospectively analyzed using public database and patient data from Wuhan Union Medical College Hospital.To investigate the effect of PLD1 on the proliferation of gastric cancer cells(SGC7901,BGC823,AGS)under nutritional stress by small molecule inhibitors.Lentivirus plasmid vector was used to construct SGC7901 cell line with PLD1 stable knockout,and the effect of PLD1 knockout on the proliferation ability of gastric cancer cell transplantation tumor was verified in vivo.The changes of mitochondrial ATP production,membrane potential and ROS after PLD1 down-regulation were analyzed by flow cytometry.Colocalization of lipid droplets,mitochondria and autophagosomes by immunofluorescence technique was used to verify the effect of PLD1 down-regulation on lipid metabolism transformation of gastric cancer cells under nutritional stress.Through the high-throughput detection of free fatty acids,the changes of intracellular free fatty acid metabolism after PLD1 down-regulation were analyzed,and the effects of PLD1 on the PPARs pathway,as well as related nodes of its downstream regulation of free fatty acid metabolism,were verified by q RT-PCR.Western Blot was further used to verify the blocking nodes of autophagy flow by targeting PLD1,and immunofluorescence localization was used to analyze the blocking sites of autophagy flow and its mechanism.Results: Retrospective analysis results showed that PLD1 expression level was positively correlated with the prognostic risk of gastric cancer patients.MTT results showed that downregulation of PLD1 inhibited the proliferation of gastric cancer cells(SGC7901,BGC823,AGS)under conventional nutrition,and the inhibition effect was more obvious under nutritional stress and even caused death of gastric cancer cells.In vivo experimental results confirmed the growth restriction of gastric cancer cell transplantation tumor model after PLD1 down-regulation.The flow cytometry was used to detect that under nutrient stress conditions,ATP production level continued to decrease after PLD1 down-regulation,mitochondrial ROS increased in advance,but mitochondrial membrane potential was not affected,and the addition of exogenous energy substances could restore cell proliferation ability.By immunofluorescence assay,PLD1 down-regulation did not affect the contact between lipid droplets and mitochondria and the function of lipid hydrolysis,but the normal volume of lipid droplets could not be maintained after the activation of autophagy flow.Highthroughput sequencing of fatty acids showed that a large amount of free fatty acids were accumulated in the cells after the down-regulation of PLD1,and the total amount did not change much over time,indicating that fatty acid metabolism was blocked.q RT-PCR results showed that the activation of PPAR? pathway was limited,and the application of PPAR? small molecule activator could restore part of the PLD1 proliferation inhibition phenotype.Western Blot results showed that autophagy flow was blocked,but m TOR pathway was not affected.Immunofluorescence results showed that the volume of lysosome was significantly increased after PLD1 down-regulation.PLD1 and chloroquine had similar effects on lysosome,and the combination of PLD1 and chloroquine could not enhance this effect.Conclusion: The expression level of PLD1 is related to the prognosis of gastric cancer,and PLD1 activity is necessary for the maintenance of the biological behavior of gastric cancer cells.PLD1 affects the oxidation of free fatty acids by acting on the PPAR?/ ACSLS pathway.At the same time,it interferes with the function of lysosome and blocks autophagy,thus affecting the source of free fatty acids and interfering with the balance of metabolism of free fatty acids,thus reducing the metabolic plasticity of cells. |
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