The Mechanism Of CYP2S1 Affecting The Proliferation,Invasion And Migration In Lung Adenocarcinoma

Objective(s): Lung cancer is a malignant tumor with high incidence and mortality worldwide,and adenocarcinoma accounts for the highest proportion of pathological types of lung cancer and is on the rise.Because lung adenocarcinoma has no typical clinical manifestations in the early stage,and blood tract and lymph node metastasis can occur when the primary lesion is small,most patients are already in the middle and advanced stages at the time of presentation,and the clinical treatment effect is poor.Therefore,exploring the biomarkers of lung adenocarcinoma is of great significance for the early diagnosis and treatment of patients.Previous studies on cytochrome P450(CYP450)family monooxygenases have focused on their catalytic oxidation of organic compounds,and their role and mechanism in malignant tumors have been poorly studied.This study aimed to investigate the relationship between CYP2S1 of CYP450 family and the survival prognosis of lung adenocarcinoma patients,its effect on the biological function of lung adenocarcinoma cells,and to further explore its mechanism of action causing proliferation and invasion migration of lung adenocarcinoma cells.prognosis of lung adenocarcinoma patients1.To detect the effect of differential expression of CYP2S1 on the survival1.1 The Kaplan Meier plotter database was used to analyze the relationship between differential expression of CYP2S1 and overall survival(OS)of 3,5 and10 years and first progressive survival(FP)of lung adenocarcinoma at 3,5 and 10 years.Methods:2.To investigate the effect of CYP2S1 expression on the biological function of lung adenocarcinoma cells2.1 Western blot experiment was used to detect the basal expression level of CYP2S1 in normal bronchial epithelial cell lines,lung squamous cell carcinoma and lung adenocarcinoma cell lines;2.2 Two lung adenocarcinoma cell lines with high CYP2S1 expression were infected with sh RNA lentivirus to construct a lung adenocarcinoma model that knocked out CYP2S1,and fluorescence photography and Western blot experiments were used to detect infection efficiency and knockdown effect,respectively.2.3 CCK-8,Ed U cell proliferation experiments and plate cloning experiments were used to detect the changes in the proliferation of lung adenocarcinoma cells after knocking down CYP2S1;2.4 Flow cytometry experiments to detect the effect of knocking out CYP2S1 on the cycle of lung adenocarcinoma cells;2.5 Western blot was used to detect cell cycle-related protein changes after knocking out CYP2S1;2.6 The effect of knocking out CYP2S1 on the invasion and migration of lung adenocarcinoma cells was detected by scratch,Transwell migration and invasion experiments.2.7 Western blot detected the changes of invasion and migration-related proteins after knocking down CYP2S1;3.To explore the molecular mechanism of CYP2S1 in promoting proliferation,invasion and migration of lung adenocarcinoma3.1 The correlation between CYP2S1 expression and PPARα and GPX4 was analyzed through GEPIA online database.3.2 Western blot was used to detect the effect of knocking down CYP2S1 on the expression of downstream pathway proteins PPARα and GPX4;3.3 Effect of flow cytometry detection of knockdown CYP2S1 on ferroptosis positive detection factor ROS.Results:1.To detect the effect of differential expression of CYP2S1 on the survival prognosis of lung adenocarcinoma patients1.1 The results of Kaplan Meier plotter database analysis showed that patients with low expression of CYP2S1 had better 3,5,10-year OS and 3,5,and10-year FP than patients with high expression of CYP2S1.2.Knockout CYP2S1 inhibits the proliferation,invasion and migration ability of lung adenocarcinoma cells2.1 The results of Western blot experiments showed that compared with normal bronchial epithelial cells 16-HBE,CYP2S1 was highly expressed in lung adenocarcinoma cell lines PC-9 and A549(P<0.05),high in lung squamous cell line H226(P<0.05),and low in lung squamous cell line H1703(P<0.05);2.2 PC-9 and A549 lung adenocarcinoma cells with relatively high CYP2S1 expression were selected as experimental cells,and sh RNA lentiviral knockdown CYP2S1 was used,and the fluorescence photographic results showed that the infection rate was above 80%,and the Western blot experimental results for detecting lentiviral knock-down ability showed that compared with the Scramble group,Lentiviruses sh CYP2S1-3 and sh CYP2S1-2 significantly knocked out endogenous CYP2S1 in PC-9 and A549 cells(P<0.05).2.3 The results of CCK-8,Ed U cell proliferation experiments and plate clonalization experiments showed that the in vitro proliferation capacity of PC-9 and A549 cells was significantly inhibited after knocking down endogenous CYP2S1(P<0.05).2.4 The results of flow cytometry showed that after knocking down endogenous CYP2S1,the cycle of PC-9 and A549 cells was blocked in the G1 phase(P<0.05).2.5 The results of Western blot experiments on cycle-related proteins showed that the expression of G1-related proteins CDK4,Cyclin D1,Cyclin D2 and c-Myc in PC-9 and A549 cells was significantly reduced after knocking down endogenous CYP2S1(P<0.05).2.6 The results of cell scratching,Transwell migration and invasion showed that the in vitro migration and invasion ability of PC-9 and A549 cells were significantly inhibited after knocking out endogenous CYP2S1.2.7 Western blot assays of migration and invasion-related proteins showed that the expression of MMP9 protein in PC-9 and A549 cells was significantly reduced after knocking down endogenous CYP2S1(P <0.05).3.CYP2S1 may regulate GPX4 through PPARα to inhibit ferroptosis and promote the proliferation,migration and invasion of lung adenocarcinoma3.1 The results of GEPIA online database analysis showed that the expression of CYP2S1 in both normal and tumor tissues of lung adenocarcinoma was positively correlated with PPARα and the expression of GPX4.3.2 The results of Western blot showed that the expression of PPARα and GPX4 in PC-9 and A549 cells decreased significantly after knocking out CYP2S1(P<0.05).3.3 The results of ROS flow cytometry showed that the ROS content of the positive detection factor of ferroptosis increased significantly after knocking down CYP2S1(P<0.05).Conclusion(s):1.High expression of CYP2S1 is closely related to the poor prognosis of lung adenocarcinoma patients.2.CYP2S1 promotes proliferation,invasion and migration of lung adenocarcinoma cells.3.CYP2S1 may inhibit ferroptosis and promote the occurrence and development of lung adenocarcinoma by upregulating PPARα and GPX4.