SHMT2 Interact With Lactotransferrin To Induce Mitochondrial Injury And Ferritinophagy:to Study Ferroptosis Mechanism Of Xuanwei Lung Cancer Cancer

Objective（s）:To explore the inhibitory effect and specific mechanism of forroptosis induced by SHMT2 inhibitor SHIN1 on Xuanwei lung cancer,providing a new research direction for the treatment of Xuanwei lung cancer.Methods:SHIN1-induced ferroptosis assay:SHMT2 inhibitor SHIN1,MTHFD1/2inhibitor LY345899,Erastin,PHGDH inhibitor NCT503 and Pemetrexed treated XLA-07,XL-JT,A549 and PC-9 cells and tested survival rates.The effects of Ferrostatin-1 antioxidant NAC,Necrostatin-1 and Z-VAD-FMK on SHIN1-induced cell death were examined.The susceptibility of cells to Erastin-induced ferroptosis was detected by constructing lentivirus stable knockout SHMT2 cell lines.Lipid peroxidation induced by SHIN1 was detected by MDA levels.Mitochondrial damage induced by SHIN1:mitochondrial membrane potential,ATP level,mitochondrial ROS,and mitochondrial lipid peroxide were detected to detect the damage caused by SHIN1 to mitochondria,and FCCP and NADPH interfered with the damage caused by SHIN1 to mitochondria.Experiments on the mechanism of SHIN1 inducing ferroptosis:The interaction between SHMT2 and LF was found by CO-IP and mass spectrometry experiments and verified by CO-IP experiments.The regulation of SHIN1 on the expression levels of LF,TFR,NCOA4,Ferritin and GPX4 was detected by Western blot experiment.Mitochondrial membrane potential,ATP level and mitochondrial ROS were detected.And mitochondrial lipid peroxide to detect the regulation of silenced LF on mitochondrial function.Western blot assay was used to detect the regulation of silent LF on the expression levels of TFR,NCOA4,Ferritin and GPX4.The regulation of SHIN1,SHIN1 and 3-MA on the expression levels of LC3B-Ⅱand LC3B-Ⅰwas detected by Western blot assay.Results:The resistance of Xuanwei lung cancer cells XLA-07 and XL-JT to pemetrexel was stronger than that of common lung adenocarcinoma cells A549 and PC-9.SHIN1 inhibited the proliferation of lung adenocarcinoma cells,and Ferrostatin-1 and NAC interfered with the inhibition of SHIN1 on cell proliferation.When SHMT2 was inhibited,mitochondrial membrane potential decreased,ATP level decreased,mitochondrial ROS and lipid peroxides increased,and lipid peroxides and MDA levels increased.When SHMT2 was inhibited or silenced,LF,TFR and NCOA4 expressions were up-regulated,Ferritin expression was down-regulated,mitochondrial Fe²⁺content was increased,and cell iron content was increased.SHMT2 interacts with downstream substrate LF,silencing LF,TFR and NCOA4expression down,Ferritin expression up,mitochondrial Fe²⁺content decreased,cell iron content decreased,mitochondrial membrane potential increased,ATP content increased,mitochondrial ROS and lipid peroxide levels decreased.SHIN1 increased the transformation of LC3B-Ⅰto LC3B-Ⅱ,while 3-MA combined with SHIN1reduced the transformation of LC3B-Ⅰto LC3B-Ⅱ.Conclusion（s）:SHMT2 inhibitor SHIN1 induces ferroptosis in Xuanwei lung cancer cells.SHMT2 interacts with LF and mediates ferroptosis by regulating cellular ferritinophagy and mitochondrial damage.