Study On The Mechanism Of Genistein Inhibiting Lung Adenocarcinoma Progression Via Regulating N~6-methyladenosine(m~6A)

Objective:To explore the inhibitory effect of genistein on lung adenocarcinoma cells in vitro and in vivo,and then futher discuss the potential mechanism from the perspective of m~6A modification pathway.Methods:1.To detect the inhibitory effects of genistein on A549 and H1975 cells.The cell proliferation ability of A549 and H1975 cells was detected by MTT reduction assay.The cell migration ability of A549 and H1975 cells was detected by wound healing assay.The cell invasion ability of A549 and H1975 cells was measured by Transwell assay.2.To detect the effect of genistein on the level of m~6A in lung adenocarcinoma cells.The levels of m~6A in lung adenocarcinoma and adjacent tissues were detected by colorimetry.The levels of m~6A in normal lung epithelial cells BEAS-2B,A549 and H1975 cells were detected by colorimetry.The levels of m~6A in A549 and H1975 cells were detected by colorimetry after genistein treatment.3.To explore the effect of genistein downregulates the level of m~6A by directly targeting to METTL3.To explore the differentially expressed m~6A modifying enzyme METTL3 in lung adenocarcinoma tissues from TCGA database and verify it in clinical samples and cell lines.To construct METTL3 knockdown and overpression cell lines and detect the level of m~6A and cell proliferation,migration and invasion ability.The effect of genistein on the expression level of METTL3 in cells was detected by Western blot.4.To explore the interaction between genistein and METTL3 protein.The affinity between genistein and METTL3 protein was detected by SPR(surface plasmon resonance),and the molecular docking was studied by autodock Vina 1.1.2 software.5.METTL3 inhibited the expression of ITGB1 by regulating the level of m~6A.Genistein promoted the degradation of ITGB1 through METTL3-m~6A pathway.The target gene ITGB1 in A549 and H1975 cells was screened by transcriptome sequencing and m~6A-seq after genistein treatment.The target gene ITGB1 was verified by Me RIP and luciferase assay.The expression level of ITGB1 was detected by Western blot after genistein treatment.The stable cell lines of ITGB1 knockdown and over-expression were constructed.The effect of ITGB1 knockdown on the expression of its related pathway proteins was detected by western blot.6.To establish a xenograft tumor model,the mice were casually divided into five subgroups:model group,Genistein group(20mg/kg、40mg/kg、80mg/kg)and METTL3 knockdown group.A549 and H1975 cells were transplanted in situ to prepare nude mice lung adenocarcinoma model,and the weight was weighed every two day,and the nude mice were killed after genistein-treated 15 days.The proliferation of tumor cells in tumor tissue was detected by Proliferating cell nuclear antigen(PCNA)staining.The expression of METTL3 and ITGB1 was detected by immunohistochemistry.Results:1.Genistein significantly inhibited the proliferation,migration and invasion of A549 and H1975 cells.2.The m~6A levels of A549 and H1975 cells were significantly higher than those of normal lung epithelial cells,and the m~6A levels in A549 and H1975 cells decreased significantly after treated with genistein.3.Genistein downregulated the level of m~6A in lung adenocarcinoma cells by inhibiting the expression of METTL3.Bioinformatics analysis showed that the level of m~6A modifier METTL3 in lung adenocarcinoma was significantly higher than that in the adjacent tissues with the worse the prognosis of the patients.After treated with genistein,the expression level of METTL3 in A549 and H1975 cells was significantly reduced.The proliferation,migration and invasion ability of A549 and H1975 cells were significantly inhibited.After konckouting METTL3 in A549 and H1975 cells,the levels of m~6A decreased significantly,contradictory results have been obtained in METTL3 over-expression cells.4.Genistein directly targeted METTL3.The SPR results showed that there is a binding between genistein and METTL3 protein.The molecular docking results showed that genistein binds to the active site of METTL3 protein,and interacted with ASP 377,Cys 376,ASN 549 and SER 511 near the active site and interacted with PHE 534 formed Pi-Pi interaction,which was closely combined with each other and had better matching.5.The candidate genes were identified by the cross collection of genes with significantly lower gene expression and m~6A level.The modified gene of METTL3 was determined as ITGB1.Me RIP and dual luciferase reporter assay showed that METTL3 directly targeted ITGB1 and regulated the level of m~6A.The expression of ITGB1 was significantly reduced after treatment of genistein.The proliferation,migration and invasion of A549 and H1975cells were significantly inhibited after ITGB1 knockdown.Contradictory results have been obtained in ITGB1 over-expression cells.6.The tumor volumes in Genistein-treated groups and METTL3 knockdown group were much smaller than those in model group.PCNA staining showed that compared with the model group,the positive rates of PCNA in tumor tissue of Genistein-treated groups and METTL3 knockdown group were significantly hardly existed.The expression levels of METTL3 and ITGB1 in tumor tissue of model group were extremely high,while the expression levels of METTL3 and ITGB1 in tumor tissue of Genistein-treated groups were significantly decreased.Conclusion:Genistein can significantly inhibit the proliferation,migration and invasion ability of lung adenocarcinoma A549 and H1975 cells,and can significantly inhibit the tumor growth in vivo.The mechanism may be that genistein directly targeted METTL3,downregulated the level of m~6A in ITGB1 m RNA,and promoted the degradation of ITGB1,which performed an oncogene.