MiR-21-5p Mediates Doxorubicin-induced Apoptosis In Triple Negative Breast Cancer Via DUSP8 Regulation Of P38/MAPK Pathway

ObjectiveThe important miRNAs involved in the regulation of triple-negative breast cancer were analyzed and screened through the public cancer database,and then the biological functions of differentially expressed miRNAs and their target genes were predicted by bioinformatics.To investigate the effects of key miRNAs(miR-21-5p)of human triple negative breast cancer on the apoptosis of triple negative breast cancer cells MDA-MB-231,and to further explore the therapeutic effect of doxorubicin on triple negative breast cancer and P38 / MAPK The pathway is related,and this association is completed by miR-21-5p regulating DUSP8.Methods(1)Screening differentially expressed miRNAs in breast cancer and adj acent tissues using TCGA,GEO database and differential miRNAs screenin g and enrichment analysis of GO and KEGG pathways to identify candidat e miRNAs and target genes.(2)MTT detects the effect of ADM on the sur vival rate of MDA-MB-231 cells,and selects the optimal concentration of A DM.(3)PCR detection of miR-21-5p gene expression level in MDA-MB-231 cells after ADM treatment;detection of miR-21-5p mimics transfection effici ency to determine the optimal transfection time.(4)Cell scratch test to observe the effects of ADM,ADM+ SB203580 and overexpression/ silent miR-21-5p on MDA-MB-231 cell migration and wound healing.(5)Hoechst33258 experiment observed the morphological changes of MDA-MB-231 cell apopt osis by ADM,ADM+ SB203580 and overexpression/ silence miR-21-5p.(6)Flow cytometry was used to detect the effects of ADM,ADM+ SB203580 a nd overexpression/ silence miR-21-5p on MDA-MB-231 cell apoptosis.(7)Western blot detection of DUSP8,Bax,Bcl-2,Caspase-3,Caspase-9,P38,P-P38 in MDA-MB-231 cells by ADM,ADM+ SB203580 and overexpression/ silence miR-21-5p Protein expression level.Results一.Screening and verification of genes1.Using the TCGA database,it was found that the expression level of miR-21-5p in breast cancer tissues was significantly higher than that in adjacent tissues(log FC= 5.557,P<0.01);GEO database screening showed that miR-21-5p was in TNBC cell line The expression level was significantly increased,and the expression level of miR-21-5p was significantly reduced after chemotherapy in TNBC patients(log FC=-5.07,P<0.01);KEGG pathway enrichment and targetscan analysis showed that DUSP8 was the target of miR-21-5p The gene affects the occurrence and development of TNBC through the p38 mitogen-activated protein kinase(P38-MAPK)pathway.2.The MTT experiment found that after ADM acted on MDA-MB-231 cells,the cell survival rate decreased with the increase of the administration concentration.When the concentration was 10 μmol/L,the cell survival rate was 55.762%,tending to 50%,so ADM The best experimental concentration is 10μmol/L.3.After ADM treatment,the expression level of miR-21-5p gene in the cells decreased significantly(P< 0.001),and the expression level of DUSP8 increased significantly(P< 0.001).二.ADM induces apoptosis of MDA-MB-231 cells through the P38-MAPK pathway1.The cell scratch test found that after ADM and ADM combined with P38-MAPK pathway inhibitor(ADM+ SB203580)group intervened in MDA-MB-231 cells for 24 and 48 hours,the cell migration speed and repair ability were significantly reduced.The scratched areas were: 71.83%,67.75%,91.37%,and 84.46%;the joint action group had the strongest effect on MDA-MB-231 migration.2.Hoechst 33258 experimental observation found that after the ADM and ADM+ SB203580 group,the cell nucleus showed different degrees of debris highlighting,indicating that the cell has an apoptotic state.3.Flow cytometry found that both ADM and ADM+ SB203580 can promote the apoptosis of MDA-MB-231 cells,the apoptosis rates were 36.40% and 40.23%,respectively,and the ADM+ SB203580 group had the strongest pro-apoptotic effect(P<0.05).4.After ADM and ADM+ SB203580 intervention,the expression levels of p-p38,Pro caspase-3,Caspase-9,Bcl-2 protein in MDA-MB-231 cells were significantly reduced(P< 0.001),DUSP8,Cleaved caspase-3 The expression level of Bax protein increased significantly(P< 0.001),and the ADM+ SB203580 group had the strongest effect(P< 0.05).三.miR-21-5p affects MDA-MB-231 cell apoptosis through the DUSP8-P38/ MAPK pathway1.After transfection of miR-21-5p mimics and inhibitor with MDA-MB-231 cells,the transfection efficiency is highest at 24 h.2.24 hours after transfection with miR-21-5p mimics and inhibitor,the miR-21-5p mimics group increased cell migration speed and repair ability(P< 0.05),and the miR-21-5p inhibitor group cell migration ability was significantly reduced(P< 0.001).3.24 hours after transfection,the apoptosis rate of miR-21-5p inhibitor group was 36.14%,which was significantly higher than that of normal group(P< 0.001).The apoptosis rate of miR-21-5p mimics group was the same as that of normal group The difference is not statistically significant(P> 0.05)4.24 hours after transfection,the expression levels of p-p38,Pro Caspase-3,Caspase-9,Bcl-2 protein in miR-21-5p mimics group were significantly increased,while the expression levels of DUSP8,Cleared Caspase-3,Bax protein were significantly reduced(P< 0.001),miR-21-5p inhibitor protein expression level was opposite to the mimics group.Conclusions1.miR-21-5p as an oncogene affects the occurrence and development of TNBC.2.ADM inhibits the expression of miR-21-5p,and then activates the expression of DUSP8,which further affects the expression of P38/MAPK and apoptosis-related proteins,and ultimately promotes the apoptosis of MDA-MB-231.