The Inhibition Mechanism Of OP-B On Angiogenesis,Migration And Invasion In NSCLC

ObjectiveThe aim of this study was to clarify the inhibitory effect of ophiopogon B(OP-B)on the migration,invasion and angiogenesis of non-small cell lung cancer(NSCLC)A549 and H460 cells,as well as the molecular mechanism by which OP-B inhibits the migration and invasion of A549 cells in the inflammatory microenvironment,so as to provide a molecular basis of OP-B anti-NSCLC,and to give a new understanding of the anticancer mechanism of OP-B on NSCLC.MethodsIn the first part,transwell assay,scratch assay and western blot were used to verify the effect of OP-B on migration/invasion and the EMT pathway in A549 cells.In vivo,by pulmonary metastasis nude mouse model and HE stained tumor sections to verify the anti-NSCLC migration and invasion effect of OP-B.Otherwise,matrigel assay analysis was used to detect microtubule formation in EA.hy926 cells after treatment with OP-B,and the western blot experiment was used to detect the vascular-associated proteins in A549 cells.In vivo,matrigel plug assay was used to detect the effect of OP-B on angiogenesis and hemoglobin in A549 transplanted tumor.In the second part,the inflammatory microenvironment was simulated by co-culturing A549 cells with LPS-treated THP-1 cells,transwell assay and western blot were used to verify the effect of OP-B on the invasion and migration in A549 cells.The qRT-PCR technique was used to detect the effect of OP-B on linc00668 expression in A549 cells in inflammatory.Bioinformatic prediction and dual-luciferase reporter gene assay verify the linc00668 sponge miR-432-5p,qRT-PCR technique was used to detect the effect of OP-B on the expression level of miR-432-5p in A549 cells and the expression of E-cadherin,N-cadherin and Vimenten after overexpression of miR-432-5p.Westem blot analysis of EMT-related protein expression in A549 cells transfected with linc00668 or overexpressed with miR-432-5p plasmid.ResultsThe first part:Screening of transcriptome and digital gene expression(DGE)profling data in NSCLC cell lines showed that OP-B regulated the epithelial-mesenchymal transition(EMT)pathway in A549 cells.Further results showed that 10?mol/L OP-B downregulated EphA2 expression and phosphorylation(Ser897)in A549 cells but upregulated them in H460 cells.Meanwhile,the Ras/ERK pathway was unaffected in A549 cells and stimulated in H460 cells.More importantly,detection of the EMT pathway showed that OP-B treatment increased the epithelial markers ZO-1 and E-cadherin and decreased the expression of the mesenchymal marker N-cadherin and the transcriptional repressors Snail,Slug and ZEB1.Furthermore,through transwell migration and scratch wound healing assays,we found that 10?mol/L OP-B significantly reduced the invasion and migration of A549 cells.In vivo,we found that 75 mg/kg OP-B inhibited A549 cell metastasis in a pulmonary metastasis nude mouse model.In addition,we also found that 10 ?mol/L OP-B significantly inhibited tube formation in EA.hy926 cells.The expression of VEGFR2 and Tie-2,the phosphorylation of Akt(S473)and PLC(S1248),and the levels of EphA2 and phosphorylated EphA2(S897)were all inhibited by OP-B in this cell line.In vivo,using a Matrigel plug assay,we found that OP-B inhibited angiogenesis and the hemoglobin content of A549 transplanted tumors.The second part:Digital Gene Expression(DGE)analysis of non-small cell lung cancer(NSCLC)cell lines showed that OP-B downregulated the expression of linc00668,which altered progression of cancer.Herein,we simulated the inflammatory microenvironment by co-culturing A549 cells with LPS-treated THP-1 cells and found that the level of linc00668 increased significantly in the mock group,while OP-B treatment inhibited the level of linc00668 and reversed epithelial-mesenchymal transition(EMT)induced by linc00668.In addition,overexpression of linc00668 in A549 cells suppressed the expression of E-cadherin and induced expression of N-cadherin,while OP-B treatment reversed these changes.Bioinformatic prediction and dual-luciferase reporter gene assay validated that linc00668 sponge miR-432-5p and at last acted on EMT to execute the anti-migration function of A549 cells under inflammatory microenvironment.Conclusions1.OP-B suppresses metastasis and angiogenesis of adenocarcinoma A549 cells by inhibiting EphA2/Akt and the corresponding pathway in vitro and in vivo.2.In inflammatory microenvironment OP-B inhibits metastasis of A549 cells via the linc00668/miR-432-5p/EMT axis.