MiR-489Regulates Chemoresistance In Breast Cancer Via Epithelial Mesenchymal Transition Pathway

Chemotherapy is frequently used for breast cancer treatment, but the major impedimentto success is the development of chemoresistance. Various factors may cause chemoresistance,including genetic changes and epigenetic changes.miRNAs play an important inchemoresistance. We investigated the role of miRNAs in the development of resistance toAdriamycin.Our study used human breast cancer (MCF-7/WT) cells and Adriamycin(ADM)-resistant (MCF-7/ADM) cells to investigate the dysregualtion miRNAs, we examinedthe global miRNA expression profiles (unpublished data) and identified miR-489was one ofthe most markedly changed miRNAs. We then used real-time PCR and in situ hybridization(ISH) to validate the expression of miR-489; this showed that miR-489was significantlydown-regulated in MCF-7/ADM cells compared to MCF-7/WT cells. MTT assay showed thatafter agomir-489transfection, MCF-7/ADM cells became more sensitive to ADM-inducedcell death.In a search for potential targets of miRNA-489that might be involved in the resistance toADM and the EMT-like phenotype in MCF-7/ADM cells, we used TargetScan software topredicte the potential targets. HEK293T cells were then transfected with miR-489togetherwith the luciferase reporter vector and the luciferase activity was assessed. The resultsdemonstrated that miR-489suppressed the expression of luciferase and thus targeted the3’UTR of Smad3. We then examined the interaction between Smad3and miR-489inMCF-7/ADM cells. Western blot results showed that Smad3was up-regulated inMCF-7/ADM cells compared to MCF-7/WT cells, while forced expression of miR-489inMCF-7/ADM cells significantly decreased Smad3expression by immunofluorescencestaining and western blot.Because Smad3plays an important role in EMT, so we study weather MCF-7/WT andMCF-7/ADM cells possessed EMT property. We found that MCF-7/ADM cells changes inmolecular markers, displayed mesenchymal-like markers, such as the high expression ofvimentin, the absence of E-cadherin, suggesting EMT-like features. In addition, transwellresults showed that MCF-7/ADM cells have high migratory ability compared to MCF-7/WTcells.We then investigated whether the down-regulation of miR-489was involved in thegaining of EMT properties. For this purpose, MCF-7/ADM cells were transfected withagomir-489, and we used reverse transcript PCR, immunofluorescence staining and westernblot assays to test the expression of E-cadherin and Vimentin. The results showed thatrestoring the activity of miR-489decreased the expression of Vimentin and increased theexpression of E-cadherin at the RNA and protein levels. As a result, miR-489significantlyinhibited the migration of MCF-7/ADM cells.We further assessed gene/miRNA expression in nude mice xenograft tumors and clinicalsamples. Immunohistochemistry showed low E-cadherin and abundant Vimentin and Smad3 in MCF-7/ADM xenograft tumors compared with MCF-7/WT tumors. In situ hybridizationassays showed that miR-489expression was lower in MCF-7/ADM than in MCF-7/WTxenograft tumors. To further verify our results, we assessed the expression and correlation ofmiR-489, Smad3, E-cadherin, and Vimentin in clinical samples of breast cancers.Immunohistochemistry showed that miR-489was down-regulated, while Smad3wasup-regulated in non-responders compared to responders. At the same time, we found that lowmiR-489expression and high-Smad3expression were highly correlated with the EMTfeatures of low E-cadherin and high Vimentin expression in the samples from non-responders.We are the first to report that loss of miR-489/gain of Smad3is a potential modulator ofboth chemoresistance and EMT-like properties in breast cancers. Therefore, re-expression ofmiR-489or inhibition of Smad3might be potential therapeutic approaches for the treatmentof chemoresistant breast cancers.