Downregulation of exosomal miR-7-5p promotes breast cancer migration and invasion by targeting RYK and participating in the atypical WNT signalling pathway

Abstract

Background: Current studies show that exosomal miRNAs become an important factor in cancer metastasis. Among the many miRNA studies, miR-7-5p has not been thoroughly investigated in breast cancer metastasis.

Methods: Bioinformatic screening was performed using extant data from the GEO database, and miR-7-5p expression levels in breast cancer cell lines and exosomes were further examined using real-time quantitative PCR (qRT-PCR). The extracted exosomes were characterised by transmission electron microscopy (TEM), particle size analysis and marker protein determination. Cell migration and invasion were then examined using wound healing assays and Transwell assays, respectively. Correlation between miR-7-5p and receptor-like tyrosine kinase (RYK) was analysed by luciferase reporter. The effect of miR-7-5p against RYK-related downstream factors was verified using western blot assays.

Results: In this study, we found that the expression of miR-7-5p was significantly different in exosomes secreted from breast cancer cell lines with different high and low invasiveness. Further experiments revealed that miR-7-5p has an important role in inhibiting the migration and invasion of breast cancer. In terms of mechanism of action, miR-7-5p was found to target the RYK, leading to its reduced expression, which in turn caused a reduction in the phosphorylation level of the downstream factor JNK. Reduced levels of phosphorylated JNK factors lead to reduced levels of phosphorylation of c-Jun protein, which in turn leads to increased expression of EMT transcription factors, thereby inhibiting the epithelial-mesenchymal transition (EMT) process to suppress the invasion of breast cancer.

Conclusion: Thus, we demonstrated that exosomes loaded with high levels of miR-7-5p emitted from less aggressive breast cancers can participate in the atypical WNT pathway by targeting the RYK gene and thus inhibit breast cancer metastasis.

Keywords: Atypical WNT pathway; Breast cancer; EMT; Exosome; RYK; miR-7-5p.

Fig. 1

Exosomes from highly invasive breast cancers are more likely to promote cancer cell migration than less invasive ones. A Transmission electron microscopy of exosomes extracted from MDA-MB-231 and MCF7 breast cancer cell lines. Scale bar, 200 nm. B Particle size distribution of isolated exosomes. C Western blot analysis of Alix, TSG101 and CD63, marker proteins of exosomes isolated from MDA-MB-231 and MCF7 cells. D Exosomes were labelled using Dio stain and co-incubated with MCF7 and MDA-MB-231 cells for 3 h. Scale bar, 10 μm. E Wound healing experiments with MCF7 and MDA-MB-231 cells treated with MDA-MB-231 exosomes and MCF7 exosomes, respectively. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Bioinformatic analysis to screen for differentially expressed miRNAs. A Bioinformatic analysis of the volcano plot results showed that MDA-MB-231 and MCF7 source differentially expressed miRNAs in exosomes. B The expression of selected eight up-regulated miRNAs in exosomes and cells was examined using real-time quantitative PCR. C Real-time quantitative PCR was used to detect the expression of selected five down-regulated miRNAs in exosomes and cells, respectively. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Downregulation of exosomal miR-7-5p expression significantly facilitates the migration and invasion of breast cancer cells. A Expression of miR-7-5p in the secreted exosomes of blank control, miR-7-5p mimic and suppressor transfected MDA-MB-231 cells was measured using qRT-PCR assay. B, C The expression of miR-7-5p in MDA-MB-231 cells and MCF7 cells after transfection of blank control, miR-7-5p mimics and inhibitors and their negative controls was measured using qRT-PCR assay. D, E Wound healing assay using blank control, miR-7-5p mimic and inhibitor and its negative control after treatment of MDA-MB-231 cells and MCF7 cells. F, G The migratory invasion ability of cells after treatment of MDA-MB-231 cells and MCF7 cells with blank control, miR-7-5p mimics and inhibitors and their negative control was examined using Transwell assay. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Fig.4

Hsa-miR-7-5p targets the RYK gene. A The wild type (WT) and mutant (MUT) of miR-7-5p at the binding site of the 3′ UTR of the RYK gene. B Validation of miR-7-5p targeting to RYK using luciferase reporter gene assays. MDA-MB-231 cells were co-transfected with negative control (NC) or miR-7-5p mimics and luciferase reporter plasmids (including RYK 3′ UTR WT or RYK 3′ UTR MUT). C Expression of RYK mRNA in MDA-MB-231 cells transfected with NC or miR-7-5p mimics and inhibitors was measured by quantitative real-time PCR. D Western blot analysis of RYK protein expression in MDA-MB-231 cells transfected with NC or miR-7-5p mimics and inhibitors. E Wound healing experiments using blank control, siRYK and siRYK + miR-7-5p inhibitor after co-treatment of MDA-MB-231 cells. F A Transwell assay was used to verify the migratory invasion ability of MDA-MB-231 cells treated with blank control, siRYK and siRYK + miR-7-5p inhibitor. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Hsa-miR-7-5p is involved in the JNK-mediated atypical WNT signalling pathway and significantly inhibits breast cancer metastasis. A Western blot analysis of E-cadherin and N-cadherin protein expression levels after treatment of MDA-MB-231 cells with blank controls or miR-7-5p mimics, inhibitors and their negative controls. B Western blot analysis of p-JNK, p-c-Jun and ZEB1 protein expression levels after treatment of MDA-MB-231 cells with blank control or miR-7-5p mimics, inhibitors and their negative controls. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001