| Objective:Aerobic glycolysis is a unique method of obtaining energy for cancer cells.In recent years,the treatment of targeted tumor glycolysis has received widespread attention.The tumor oxidative stress microenvironment and glycolysis are connected in tandem.NADPH oxidase(Nicotinamide adenine dinucleotide phosphate oxidase,NADPH oxidase,NOX)is one of the important sources of reactive oxygen species(ROS)in tumor cells,and NADPH oxidase 4(NOX4)is a subtype of NOX.Studies have confirmed that NOX4 affects glycolysis of lung adenocarcinoma through the Akt signaling pathway,but the upstream mechanism of NOX4 regulating glycolysis in lung adenocarcinoma is currently unclear.BACH1 is highly expressed in various malignancies and can affect the production of ROS in tumor cells.Therefore,it is urgent to explore the regulatory relationship between BACH1 and NOX4,and which affects the mechanism of glycolysis in lung adenocarcinoma.Methods:In this study,the expression of BACH1 and the prognosis outcomes in lung adenocarcinoma were analyzed by sequencing information from the TCGA database.qRT-PCR and Western Blot were used to detect BACH1 expression in human lung adenocarcinoma cell lines H1299,A549,and human normal bronchial epithelial cell(HBE).A series of bioinformatics databases were used to predict the interactions among BACH1,miR-92b-3p and NOX4,and this prediction was confirmed by in vitro and in vivo experiments.The expression of BACH1 was downregulated by transfecting si-RNA into H1299 and A549,the proliferation ability of cells after BACH1 knockdown was detected by CCK8 and EdU assays,and the migration ability of cells after BACH1 knockdown was detected by Transwell assay.The changes in glycolysis after BACH1 knockdown were evaluated using ATP production,18F-FDG uptake,and lactate production assays.The expression of NOX4 and key glycolytic enzymes(GLUT1,HK2,PKM2,and LDHA)after BACH1 knockdown were detected by Western Blot,and the changes of cellular ROS after BACH1 knockdown were detected by reactive oxygen species kit.Bioinformatics was used to predict the targeted binding site between BACH1 and miR-92b-3p.The expression level of miR-92b-3p after BACH1 knockdown was detected by qRT-PCR.The binding of BACH1 to the miR-92b-3p promoter was verified by chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.Bioinformatics was used to predict the targeted binding site between miR-92b-3p and NOX4.miR-92b-3p inhibitor and miR-92b-3p mimic were transfected into H1299 and A549,and NOX4 expression levels were detected with qRT-PCR and Western Blot.The dual luciferase reporter assay verified that miR-92b-3p binds to the 3’-UTR region of NOX4.miR-92b-3p inhibitor and si-NOX4,miR-92b-3p mimic and oe-NOX4 were co-transfected into H1299 and A549,respectively.Using rescue experiments verified that miR-92b-3p affects proliferation,migration and glycolysis of lung adenocarcinoma by regulating NOX4.Finally,A549 cells treated with sh-NC and sh-BACH1 were injected into the right axilla of nude mice to establish a subcutaneous transplanted tumor model,and the expression levels of BACH1,NOX4,and glycolytic enzymes in transplanted tumor tissues were detected by immunohistochemical staining.MicroPET imaging was performed on subcutaneous transplanted tumors to verify the effect of BACH 1 on glycolysis in imagingResults:1.Sequencing information from the TCGA database showed that BACH1 was highly expressed in lung adenocarcinoma,and patients with high BACH 1 expression had shorter survival times.The expression of BACH1 in H1299,A549,and HBE was detected by qRT-PCR and Western Blot,and the results showed that the expression level of BACH1 in H1299 and A549 was higher than that of HBE in both mRNA and protein level.2.CCK8 and EdU assays were used to detect the proliferation of H1299 and A549 cells,Transwell detected cell migration,ATP production,18F-FDG uptake rate and lactate production assays to detect cellular glycolysis,and reactive oxygen species kit to detect cell ROS production levels,the results showed that the proliferation,migration,glycolysis and ROS production levels of cells were reduced after knocking down BACH1.3.Bioinformatics indicated that BACH1 binds to the promoter region of miR-92b-3p.The expression level of miR-92b-3p was detected by qRT-PCR after BACH1 knockdown,and the results showed that the expression level of miR-92b-3p in the knockdown group was increased.Five pairs of ChIP-qPCR primers were designed based on the binding sites sequence between BACH1 and miR-92b-3p predicted in the JASPAR database,two of which contained specific binding regions and the remaining three pairs were non-specific primers.In H1299 cells,the purified DNA fragments were collected by ChIP assay for q-PCR detection,and the results showed that BACH1 could bind to the promoter region of miR-92b-3p,and the binding site sequence was AGTGAGGCAGCGA.The binding sites sequence between BACH1 and miR-92b-3p was further verified by dual luciferase reporter assays.4.Bioinformatics indicated that miR-92b-3p binds to 3’-UTR of NOX4.miR-92b-3p inhibitor and miR-92b-3p mimic were transfected into H1299 and A549 cell lines,and qRT-PCR and Western Blot were used to detect the changes of NOX4 expression,and the results showed that the expression of NOX4 increased after miR-92b-3p knockdown,and the expression of NOX4 decreased after miR-92b-3p overexpression.5.In H1299 and A549 cells,miR-92b-3p inhibitor+si-NOX4 and miR-92b-3p mimic+oe-NOX4,respectively.In H1299 and A549 cells,miR-92b-3p inhibitor+si-NOX4 and miR-92b-3p mimic+oe-NOX4,respectively.The results showed that si-NOX4 could restore miR-92b-3p inhibitor on the proliferation,migration,glycolysis and ROS production level of H1299 cell line and oe-NOX4 could restore miR-92b-3p mimic on the level ability of A549 cell line proliferation,migration,glycolysis and ROS production.6.To establish a xenograft tumor model,and the nude mice were randomly divided into two groups,with nude mice injected with sh-BACH1-treated A549 cells as the experimental group and nude mice injected with sh-NC-treated A549 cells as the control group.MicroPET imaging was performed on nude mice,and the results showed that the SUVMAX of 18F-FDG uptake and growth rate in the experimental group was significantly inhibited.The expression of BACH1,NOX4,and glycolytic enzymes were detected by immunohistochemistry staining,which showed that the expression of NOX4 and glycolytic enzymes were inhibited in the experimental group compared with the control group.Conclusion:1.BACH1 is highly expressed in lung adenocarcinoma cell lines and can affect their glycolysis and migration.2.BACH1 regulates the expression of NOX4 by targeting miR-92b-3p,thereby promoting glycolysis and migration of lung adenocarcinoma cell lines. |
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