Proliferation And Migration Of Lung Cancer Could Be Inhibited By Oxymatrine Through The Regulation For MiR-520/VEGF

Background and objects:Lung cancer is the most fatal malignant tumor in the world with a 5-year survival rate of only 15%.For non-small cell lung cancer that accounts for more than 80% of lung cancer,standard chemotherapy is currently the most commonly used treatment,but its curative effect has reached a plateau.Oxymatrine(OMT)is an active quinazine alkaloid extracted from the dried roots,plants,and fruits of leguminous plant Sophora flavescens with ethanol and other organic solvents.OMT can not only prevent inflammation,oxidative stress,autophagy and other physiological processes to protect the body's tissues and organs,and also has an excellent anti-cancer effect,but the detailed mechanism of its anti-cancer activity is still not clear.Therefore,this study aims to explore the effect and potential mechanism of OMT against lung cancer.Methods:1.Using miRNA microarray to detect miRNAs expressed in control A549 cells and A549 cells treated with OMT(1.0 mg/m L)for 72 h.2.Online database predicted the target genes of the differential miRNAs,sort out and analyze the biological functions of the predicted target genes and the pathways involved.3.The expression levels of miR-520 and VEGF in lung cancer tissues and adjacent tissues in 20 cases were detected using qRT-PCR,Western blot and immunohistochemistry.4.The expression levels of miR-520 and VEGF in in H69 and A549 after different doses of OMT treatment were detected using qRT-PCR and Western blot.5.Colony formation assay and wound healing assay were performed to examine the proliferation ability of A549 cells after different doses of OMT treatment.6.Identify the interaction between miR-520 and VEGF using the dual luciferase reporter assay.7.Analyze the biological functions of proliferation,migration and invasion of lung cancer cells transfected with miR-520 mimics through MTT,Transwell,colony formation and wound healing assayes.8.Western blot detected the biomarker expression level of tumor cell migration and invasion.9.To detect the tumorigenic ability of A549 cells with higher miR-520 expression level by nude mice in vivo tumorigenesis experiment.Result:1.A total of 770 miRNAs were detected by miRNA microarray,and when the fold change was set to 2,a total of 55 differentially expressed miRNAs were screened,of which 43 were up-regulated miRNAs and 12 were down-regulated miRNAs.2.Through the prediction and analysis of the online database,the five signal pathways most likely to play a role in OMT treatment of A549 cells are: vascular endothelial growth factor(VEGF)signal pathway,interleukin signal,and cadherin signal pathway,apoptosis signaling pathway and fibroblast growth factor signaling pathway.3.qRT-PCR,Western blot and immunochemistry results showed that the expression of miR-520 in lung cancer tissue is significantly decreased and the transcription level and protein expression level of vascular endothelial growth factor(VEGF)are significantly increased,and the expression of miR-520 and VEGF showed a significant negative correlation trend in lung cancer tissues.4.qRT-PCR and Western blot results showed that the expression level of miR-520 in H69 cells and A549 cells after OMT treatment was increased,and the transcription and translation level of VEGF were decreased.This difference was significant at high doses of OMT(1.0,1.5 mg/m L)treatment.5.The results colony formation assay and wound healing assay showed that the colony formation ability and wound healing ability of A549 cells after OMT treatment were significantly reduced.6.The results of the dual luciferase assay indicate that miR-520 can directly target VEGF by binding to the VEGF 3 'UTR region,proving that VEGF is a direct target gene of miR-520.7.MTT,Transwell,colonies formation,and wound healing assays proved that overexpressed miR-520 inhibited the proliferation,migration,invasion,colonies formation and wound healing of lung cancer cells.8.Western blot experiments show that high miR-520 expression level significantly decreased the expression levels of the matrix metalloproteinases MMP-2 and MMP-9,which are the biomarkers of the ability of migration and invasion.9.The tumorigenic ability of A549 cells with high miR-520 expression level was significantly lower than that of the control group,indicating that miR-520 reduced the tumorigenic ability of A549 cellsConclusion:In summary,the increased expression of miR-520 can inhibit the proliferation,migration and invasion of lung cancer cells,reduce the tumorigenicity of A549 cells,and have a better prognosis in lung cancer patients with higher expression levels of miR-520.Mechanically,the treatment of OMT can up-regulate the expression of miR-520,and theincreased miR-520 can inhibit the expression of VEGF,and OMT treatment thereby inhibit the development of lung cancer.