| Objective:Acquisition of resistance to adriamycin(ADR)is one of the most important clinical obstacles in the treatment of breast cancer,but the molecular mechanisms underlying sensitivity to ADR remain elusive.In our previous study,through miRNA microarray and experiments,we have emphasized that miR-222 could promote the ADR-resistance in breast cancer cells.The aim of this study was to explore the possible mechanism by which miR-222 affects sensitivity to ADR.Methods:1.ADR-resistant MCF-7 breast cancer cell subline(MCF-7/ADR)was successfully established in vitro through a stepwise increase of ADR concentrations in the culture based on parental MCF-7 cell lines(MCF-7/S).MCF-7/S cells were transfected with miR-222 mimics,and MCF-7/ADR cells were transfected with its inhibitors.2.We used TargetScan and PicTar(target prediction algorithms)in conjunction with pathway enrichment analyses to predict the mRNAs that were most likely to involve in miR-222-mediated drug resistance in cancers.RT-qPCR analyses and Western Blot assays confirmed the relationship between miR-222 expression and target gene FOXO1.Immmunofluorescence further visually displayed the location of FOXO1.3.In order to further explore the mechanism,the expression levels of PTEN and Akt were detected by RT-qPCR and Western Blot,respectively.4.To testify whether the levels of miR-222 were associated with the sensitivity of breast cancer cells to ADR,flow cytometry analysis and MTT assay were also applied.5.After blocking PTEN/Akt/FOXO1 signaling pathway by LY294002(an inhibitor of Akt signaling),the levels of target genes were determined by RT-qPCR and Western Blot.And the effects of miR-222-mediated ADR resistance were demonstrated again by MTT and flow cytometry assays.6.The relationship between miR-222 and overall survival was analyzed by TCGA database.Results1.The potential target genes of miR-222 were predicted by TargetScan and PicTar,respectively.Furthermore,we narrowed the predicted target genes via KEGG pathway enrichment analysis,and they were involved in 22 pathways(P<0.05).In particular,PTEN and FOXO1 are two key members in a major sub-pathway of"hsa05200:pathways in cancer".RT-qPCR and Western Blot results showed that miR-222 expression was negatively correlated with FOXO1 expression.In addition,the subcellular translocation of FOXO1 due to the altered expression of miR-222 was observed from immunofluorescence.2.Upregulation of miR-222 expression in MCF-7/S cells is associated with decreased PTEN expression levels and increased phospho-Akt(p-Akt)expression.Conversely in MCF-7/ADR cells,inhibition of miR-222 resulted in increased PTEN expression and decreased p-Akt expression.3.Compared with negative control,after transfection with miR-222 mimics into MCF-7/S cells,the apoptotic cell numbers were significantly reduced and the IC50 value of ADR measured by MTT assay for cells was obviously increased.In contrast,after miR-222 inhibitors transfected into MCF-7/ADR cells,the apoptotic cell numbers were significantly increased and the IC50 value of ADR was dramatically reduced.4.Compared with the control,treatment with LY294002 inhibited Akt phosphorylation in MCF-7/S cells,causing increased FOXO1 expression.Additionally,LY294002 partially increased sensitivity of MCF-7/S cells to ADR,as shown by the increased apoptotic rate and decreased IC50 values.5.High expression of miR-222 is closely related to poor overall survival by TCGA database validation.Conclusions1.The expression levels of miR-222 were positively correlated with the drug resistance of breast cancer cells to ADR.2.Bioinformatics showed PTEN and FOXO1 were both specific target genes of miR-222.And their expression was negatively regulated by miR-222.3.MiR-222 could regulate the expression and subcellular localization of FOXO1 in MCF-7 cells by targeting PTEN,thereby modulating drug resistance of breast cancer cells to ADR.4.Inhibition of miR-222 predicts a relatively good prognosis of breast cancer patients. |
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