Exosomes from tamoxifen-resistant breast cancer cells transmit drug resistance partly by delivering miR-9-5p

Abstract

Background: Resistance to drug therapy is a major impediment for successful treatment of patients suffering from breast cancer (BC). Tamoxifen (TAM) is an extensively used therapeutic agent, which substantially reduces the risk of recurrence and associated mortality in BC. This study demonstrated that exosomal transfer of microRNA-9-5p (miR-9-5p) enhanced the resistance of MCF-7 cells to TAM.

Methods: Initially, BC-related differentially expressed genes (DEGs) and their upstream regulatory miRNAs were identified. The TAM-resistant MCF-7 (MCF-7/TAM) cell line and the non-medicated sensitive MCF-7 cell line were formulated, followed by isolation of the exosomes. Next, the apoptosis rate of exosome-treated MCF-7 cells was determined after co-culture with TAM. The interaction between miR-9-5p and ADIPOQ was identified by a combination of bioinformatic analysis and luciferase activity assay. In order to validate the effect of miR-9-5p and ADIPOQ on TAM resistance in the MCF-7 cells in vitro and in vivo, miR-9-5p was delivered into the exosomes. ADIPOQ and miR-9-5p were identified as the BC-related DEG and upstream regulatory miRNA.

Results: Exosomes derived from the MCF-7/TAM cells could increase the resistance of MCF-7 cells to TAM. Notably, miR-9-5p altered the sensitivity of BC cells to TAM. In addition, ADIPOQ was negatively regulated by miR-9-5p. Furthermore, MCF-7/TAM cell-derived miR-9-5p inhibited the apoptosis of MCF-7 cells, and promoted the cell resistance to TAM. In vivo experiments in nude mice ascertained that the tumor injected with exosomal miR-9-5p showed improved resistance to TAM.

Conclusions: Exosomal transfer of miR-9-5p augmented the drug resistance of BC cells to TAM by down-regulating ADIPOQ, suggesting its functionality as a candidate molecular target for the management of BC.

Keywords: ADIPOQ; Breast cancer; Drug resistance; Exosomes; MCF-7; MCF-7/TAM; MicroRNA-9-5p; Tamoxifen.

Fig. 1

Exosomes from MCF-7/TAM cells can be transferred into the parental MCF-7 cells. a TEM observation of exosomes (scale bar: 200 nm). b Nanoparticle tracking analysis of exosome concentration and size. c The expression patterns of marker proteins (CD63 and TSG101) of exosomes in MCF-7-exo and MCF-7/TAM-exo detected by Western blot analysis (normalized to β-actin). d MCF-7-exo and MCF-7/TAM-exo (labeled with PKH67 dye) could be internationalized by parental MCF-7 cells (scale bar: 25 μm)

Fig. 2

Exosomes from MCF-7/TAM cells confers drug resistance in the parental MCF-7 cells. a The cell viability of MCF-7 cells after TAM treatment determined by CCK-8. b IC₅₀ values of MCF-7 cells treated with TAM. c The cell cycle distribution of MCF-7 in the presence of MCF-7/TAM-exo, analyzed by flow cytometry. d The apoptosis of MCF-7 cells in the presence of MCF-7/TAM-exo, analyzed by flow cytometry. e Western blot analysis of stemness markers KIT, CD44, and CD24 in MCF-7 cells in the presence of MCF-7/TAM-exo. f Sphere-forming abilities of MCF-7 cells in the presence of MCF-7/TAM-exo were examined by 3-D cell culture (scale bar: 50 μm). *p <0.05. All experiments were conducted three times independently

Fig. 3

ADIPOQ and miR-9-5p is involved in the drug resistance of BC. a Heat map of BC-related DEGs in different expression profiles. The x-axis indicates the sample number, the y-axis indicates the gene name, and the left dendrogram indicates the gene expression cluster. Each small square in the panel indicates the expression of a gene in one sample. b Enrichment analysis of DEGs in KEGG metabolic pathways. The x-axis refers to GeneRatio, the y-axis refers to KEGG signaling pathways, and the right histogram represents color gradation. c Protein–protein interaction network of the DEG in the PPAR signaling pathway. Each circle represents a gene and the circle color represents the core degree of the DEG in the interaction network. Darker color means higher core degree. d Prediction of upstream regulatory miRNAs of ADIPOQ. Four ellipses refer to the results predicted from the four databases and the central section refers to the intersecting of the prediction results

Fig. 4

miR-9-5p targets and negatively regulates ADIPOQ. a The prediction of binding sites between miR-9-5p and ADIPOQ. b Quantitation of the luciferase activity assay. c The expression pattern of miR-9-5p in each group measured by RT-qPCR. d The mRNA expression pattern of ADIPOQ in the cells of each group measured by RT-qPCR. e The protein expression pattern of ADIPOQ in cells of each group evaluated by Western blot analysis. *p <0.05. Each experiment was conducted three times independently

Fig. 5

MiR-9-5p promotes TAM resistance in the parental MCF-7 cells. a The miR-9-5p expression in MCF-7 and MCF-7/TAM cell lines measured by RT-qPCR. b The cell viability of cells in response to miR-9-5p mimic or miR-9-5p inhibitor determined by CCK-8. c Quantitation of IC₅₀ value of cells in response to miR-9-5p mimic or miR-9-5p inhibitor. d Flow cytometric detection of apoptosis rate of cells transfected with miR-9-5p mimic or miR-9-5p inhibitor. *p <0.05. Each experiment was conducted three times independently

Fig. 6

Exosomes carrying miR-9-5p down-regulate the expression pattern of ADIPOQ in BC cells. a miR-9-5p expression in (MCF-7 + miR-9-5p mimic)-exo and (MCF-7/TAM + miR-9-5p inhibitor)-exo measured by RT-qPCR. b q expression pattern of miR-9-5p in the MCF-7 recipient cells after co-culture of (MCF-7 + miR-9-5p mimic)-exo or (MCF-7/TAM + miR-9-5p inhibitor)-exo measured by RT-qPCR. c The mRNA expression pattern of ADIPOQ in the MCF-7 recipient cells after co-culture of (MCF-7 + miR-9-5p mimic)-exo or (MCF-7/TAM + miR-9-5p inhibitor)-exo measured by RT-qPCR. d The protein expression pattern of ADIPOQ in the MCF-7 cells after co-culture of (MCF-7 + miR-9-5p mimic)-exo or (MCF-7/TAM + miR-9-5p inhibitor)-exo evaluated by Western blot analysis. *p <0.05. Each experiment was conducted three times independently

Fig. 7

MiR-9-5p dictates drug resistance conferred by exosomes isolated from MCF-7/TAM cells in the parental MCF-7 cells. a The viability of MCF-7 recipient cells after co-culture with (MCF-7 + miR-9-5p mimic)-exo or (MCF-7/TAM + miR-9-5p inhibitor)-exo determined by CCK-8. b The quantitation of IC₅₀ of MCF-7 recipient cells after co-culture. c Flow cytometric detection of the apoptosis of MCF-7 recipient cells after co-culture with (MCF-7 + miR-9-5p mimic)-exo or (MCF-7/TAM + miR-9-5p inhibitor)-exo. *p <0.05. Each experiment was conducted three times independently

Fig. 8

MiR-9-5p dictates drug resistance conferred by exosomes isolated from MCF-7/TAM cells by regulating ADIPOQ in vivo. a The growth curve of tumors with multipoint injection of exosomes. b The quantitation of tumor weight changes after multipoint injection of exosomes into the tumor. c The apoptosis of tissues in different groups detected by TUNEL staining. d Protein expression of ADIPOQ in tumors of different group. e Protein expression of ADIPOQ determined by Western blot analysis. *p <0.05. n = 8

Fig. 9

The schematic representation of the molecular mechanism miR-9-5p in TAM-resistant BC cells. In TAM-resistant BC cells, miR-9-5p promotes TAM resistance by negatively regulating ADIPOQ, whereby it promotes BC cell viability and inhibits apoptosis. In addition, miR-9-5p can be packaged into the exosomes derived from TAM-resistant BC cells, and transferred into the TAM-sensitive BC cells