Endoplasmic Reticulum Stress Promotes NSCLC Resistance To Etoposide Induced Apoptosis Through The SREBP1-MRP1 Axis

Objective: Endoplasmic reticulum stress is a basic cellular stress response,which can maintain intracellular protein homeostasis under endogenous or exogenous stimuli,and contribute to tumor growth and metastasis,drug resistance,etc.Overactivation can induce apoptosis and wipe out damaged cells.This paper mainly explores why tumor cells are less sensitive to etoposide under endoplasmic reticulum stress and the specific molecular mechanism.Materials and methods: Select the human-based non-small cell lung cancer cell A549,and the H1299 cell line as the experimental object.(1)MTT detection of lung cancer cells’ sensitivity to etoposide after TG treatment.(2)etoposide induced apoptosis of lung cancer cells treated with TG was detected by flow cytometry.(3)Bioinformatics analysis and WB assay were used to detect the expression of MRP1 after TG treatment.(4)Bioinformatics was used to analyze the expression of SREBP1 in lung adenocarcinoma and its gene correlation with MRP1.(5)WB assay and oil red O staining were used to explore the effects of TG treatment on intracellular lipid levels;(6)MTT assay was used to determine whether manipulation of SREBP1 expression affected etoposide sensitivity;(7)Use WB experiments and dual-fluorescent enzymes to report genetic experimental test whether SREBP1 is regulating the expression of MRP1;(8)Application of WB experiments and dual-fluorescent enzyme report genetic experiments TG and IRE11α-XBP1 channels on SREBP1;(9)WB experiment and MTT experiment were used to verify the experimental results.Results:(1)MTT showed that TG could induce a decrease in the sensitivity of non-small cell lung cancer to etoposide.(2)Flow cytometry showed that etoposide induced apoptosis decreased after TG treatment.(3)The results of biological information show that MRP1 has higher expression in lung adenocarcinoma than normal tissue and patients’ prognosis is poor.WB experiments show that the increase in MRP1 protein expression in the cell after TG is treated;(4)Bioinformatics results showed that the expression level of SREBP1 in lung adenocarcinoma was higher than that in normal tissues and the prognosis of patients was poor.The expression of SREBP1 was positively correlated with that of MRP1.(5)WB assay showed that the expression levels of SREBP1 and lipid synthesis related proteins increased after TG treatment,and oil red O staining showed that the number of lipid droplets increased after TG treatment.(6)MTT results showed that after SREBP1 knockdown,the cell viability was lower and the sensitivity to etoposide was increased.(7)WB assay showed that the expression of MRP1 protein increased after overexpression of SREBP1 c DNA,and decreased after use of SREBP1 small interfering RNA.Double luciferase reporter gene assay showed that the activity of MRP1 promoter was increased after overexpression of SREBP1 c DNA,while the activity of MRP1 promoter was decreased after use of SREBP1 small interfering RNA.Site-specific mutation assay was used to determine the binding site of SREEBP1 on the MRP1 promoter sequence.(8)WB assay and double luciferase reporter assay showed that TG up-regulated SREBP1 expression through IRE1α/XBP1/c-Myc pathway.(9)WB and MTT experiments confirmed that endoplasmic reticulum stress can up-regulate the expression of MRP1 through IRE1α/XBP1/c-Myc/SREBP1 pathway and thus resist the killing of etoposide.Conclusion:(1)Endoplasmic reticulum stress can induce resistance to etoposide induced apoptosis in non-small cell lung cancer.(2)The IRE1α-XBP1 pathway up-regulates the expression of MRP1 through SREBP1.