Study On The Mechanisms Of Ophiopogonin-B Mediated Ferroptosis On NSCLC Cells By Regulating MiR-432-5p/AURKA

[Objective]To explore the effects and molecular mechanisms of ophiopogonin-B(OP-B)on the proliferation,migration,and invasion of non-small cell lung cancer(NSCLC)cells A549 by regulating miR-432-5p,construct a ferroptosis-related prognostic model,accurately predict the relationship between ferroptosis-related molecular targets and the prognosis of patients with NSCLC,and clarify the regulatory effect of OP-B on A549 cells ferroptosis.Multi-levels verification of the molecular mechanisms of OP-B mediating NSCLC ferroptosis and other anti-tumor effects through miR-432-5p/ferroptosis marker gene(aurora kinase A,AURKA),which provides new ideas on the anti-NSCLC mechanism research of OP-B.[Methods]In the first part,qRT-PCR was used to detect the expression of miR-432-5p in various lung cancer cell lines after OP-B administration,and to screen suitable cell lines;after miR-432-5p mimics transfected,EdU fluorescence staining and plate cell cloning experiments were used to observe that the miR-432-5p whether plays an anti proliferation role on NSCLC cells;Transwell chamber correlative experiments were used to observe miR-432-5p’s effects on NSCLC cell migration and invasion abilities;Western blotting was used to detect the effect of miR-432-5p overexpression or combined OP-B administration on the metastasis and invasion-related proteins of NSCLC cells.In the second part,We first searched the ferroptosis-related data of NSCLC patients on TCGA,including transcriptomics data and clinical characteristics information,and then packaged and imported to destination folder;the prognostic gene markers closely related to ferroptosis were screened out by Lasso regression analysis;retrieved "NSCLC" on GEO database,selected corresponding "GSE" type data to pack and organize,which was used to verify the prediction model constructed by TCGA;differentially expressed genes(DEGs)associated with ferroptosis were screened out and then used to perform KEGG analysis,the goal of which was to find out which pathways the DEGs were focused on and to explore the molecular mechanism of ferroptosis in NSCLC.In the third part,A549 cells were administered with OP-B 5 μmol·L-1 for 24 hours,and then proteomic gene sequencing was performed,and KEGG bioinformatics analysis was similarly performed to detect the protein targets regulated by OP-B and the main KEGG pathway;detection of Glutathione,malondialdehyde(MDA)and iron content in A549 cells after administration of OP-B 5 μmol·L-1;Western blot detection of ferroptosis marker genes protein changes to verify the ferroptosis induction effect of OP-B on A549cells;establish an orthotopic lung cancer model in nude mice and detect the ferroptosis inducing effect of op-b in vivo;Western blotting was performed to detect the protein expression changes of NSCLC ferroptosis marker gene AURKA under different treatments such as OP-B 5 μmol·L-1 administration,miR-432-5p overexpression,and miR-432-5p overexpression combined with OP-B administration.[Results]The first part:Compared with the control group,OP-B 5 μmol·L-1 significantly up-regulated the expression multiple of miR-432-5p in lung adenocarcinoma cell lines,and A549 cells became the research object of this experiment in vitro model;Both EdU fluorescence staining and clone formation experiments showed that miR-432-5p had a significant inhibitory effect on the proliferation of A549 cells;the results of Transwell migration and invasion experiments showed that miR-432-5p significantly inhibited the migration and invasion of A549 cells;Western blot results suggested that miR-432-5p up-regulates E-cadherin protein expression in A549 cell line,down-regulates MMP-2 protein expression,and miR-432-5p overexpression combined with OP-B 5 μmol·L-1 significantly down-regulates Snail protein.It is confirmed that OP-B inhibits the metastasis and invasion of A549 cells by regulating miR-432-5p.The second part:Bioinformatics analysis identified 12 gene markers closely related to the prognosis of NSCLC,and verified them with the GEO data set;it was determined that the differentially expressed genes(DEGs)related to ferroptosis were mainly concentrated in multiple pathways related to ferroptosis.The third part:Proteomics high-throughput sequencing analysis showed that OP-B’s regulation of A549 cells focused on the ferroptosis pathway and the arachidonic acid metabolism pathway,and OP-B significantly inhibited the ferroptosis marker gene AURKA protein expression;under the condition of OP-B μmol·L-1 concentration,the MDA and iron content in A549 cells was significantly increased,and the content of glutathione in cells was decreased,indicating that ferroptosis occurred in A549 cells;Western blot verified that OP-B inhibits the ferroptosis suppressor FTH1,FTL,GPX4 protein expression and the up-regulates the ferroptosis driver gene ACSL4,PTGS2 expression,further verified the ferroptosis-inducing effect of OP-B;vivo experiments repeatedly confirmed that OP-B can promote NSCLC ferroptosis;both OP-B administration and miR-432-5p overexpression can down-regulate AURKA protein expression,and the AURKA inhibitory effect was more significant after miR-432-5p overexpression combined with OP-B administration.[Conclusions]1.miR-432-5p could effectively inhibit the proliferation,migration,and invasion of A549 cells.Op-B can play an anti-NSCLC role by upregulating the expression of mir-432-5p in A549 cells;2.A novel ferroptosis prognostic risk prediction model was constructed for NSCLC patients,which clarified the aggregation pathway of ferroptosis-related genes in NSCLC and provided a new perspective for the treatment of NSCLC;3.OP-B could induce ferroptosis in A549 cells,and it plays an anti-NSCLC role by regulating miR-432-5p/AURKA to mediate ferroptosis in A549 cells.