Exosomal miR-361-3p promotes the viability of breast cancer cells by targeting ETV7 and BATF2 to upregulate the PAI-1/ERK pathway

Abstract

Background: Malignant progression is the major cause of poor prognosis in breast cancer (BC) patients. Plasma exosomal miRNAs have been reported to be involved in tumor progression, but their roles in BC remain unclear.

Methods: We performed plasma exosomal miRNA sequencing on 45 individuals, including healthy controls and nonmetastatic and metastatic BC patients. We examined the correlation between miRNA expression in tumor tissues and plasma exosomes in BC patients by qRT‒PCR. The effects of exosomal miR-361-3p on BC cells were determined by CellTiter-Glo, migration and wound healing assays. The target genes of miR-361-3p and downstream pathways were explored by dual-luciferase reporter assay, RNA knockdown, rescue experiments, and western blotting. We utilized murine xenograft model to further assess the impact of plasma exosomal miR-361-3p on the malignant progression of BC.

Results: We found that the expression level of plasma exosomal miR-361-3p gradually increased with malignant progression in BC patients, and the expression of miR-361-3p in plasma exosomes and BC tissues was positively correlated. Consistently, exosomal miR-361-3p enhanced the migration and proliferation of two BC cell lines, MDA-MB-231 and SK-BR-3. Furthermore, our data showed that miR-361-3p inhibited two novel target genes, ETV7 and BATF2, to activate the PAI-1/ERK pathway, leading to increased BC cell viability. Finally, the consistency of the in vivo experimental results supported that elevated plasma exosomal miR-361-3p promote the malignant progression of BC.

Conclusions: We found for the first time that plasma exosomal miR-361-3p was associated with malignant progression in BC patients. Mechanistically, exosomal miR-361-3p can enhance the migration and proliferation of BC cells by targeting the ETV7 and BATF2/PAI-1/ERK pathways. Our data suggest that plasma exosomal miR-361-3p has the potential to serve as a biomarker for predicting malignant progression in BC patients.

Keywords: Breast cancer; Exosomal miR-361-3p; Malignant progression; Migration; Proliferation.

Fig. 1

Plasma exosomal miR-361-3p is associated with malignant progression in BC patients. A Heatmaps demonstrate plasma exosomal DEMs between the control group and nonmetastatic BC patients, as well as between nonmetastatic and metastatic BC (TPM ≥ 30, P < 0.05, |log₂FC|> 0.5); Venn diagrams point out plasma exosomal miRNAs that were consistently changed; and the three images on the right side show the identification of plasma exosomes. B Box line plot showing elevated expression of plasma exosomal miR-361-3p with BC malignant progression. C Plasma exosomal miR-361-3p was positively correlated with the miR-361-3p expression level in BC tissues. D Green particles are PKH67-labeled exosomes entering BC cells, and blue are DAPI-labeled BC cell nuclei. Scale bars, 20 μm. E qRT‒PCR was performed to detect the abundance of miR-361-3p in BC cells after coculture for 72 h with exosomes. F The effect of EXO-miR-361-3p on BC cell proliferation was tested using the CellTiter-Glo kit. G–H Transwell and scratch healing assays were used to test the effect of EXO-miR-361-3p on BC cell migration, and the schematic demonstrates the time intervals of exosome loading. Scale bars, 100 μm

Fig. 2

ETV7 and BATF2 are two new targets of miR-361-3p. A The GO analysis revealed distinct pathways enriched by genes that are either downregulated or upregulated in BC cells overexpressing miR-361-3p. B Intersection of our RNA-seq results (displayed as a heatmap) and miRNA target prediction algorithms from 2 databases. C–D miR-361-3p putative targeting sites in the wild-type and mutant ETV7 3'UTR and BATF2 3'UTR, and luciferase activity assays were performed to confirm the direct binding efficiency of miR-361-3p and its targets ETV7 and BATF2. E–F Relative protein expression of ETV7 and BATF2 after coculturing BC cells with EXO-miR-361-3p/EXO-miR-NC. (*P < 0.05)

Fig. 3

ETV7 and BATF2 negatively regulate the PAI-1/ERK pathway as well as the proliferation and migration of BC cells. A Relative protein expression of PAI-1, p-ERK and T-ERK after coculturing BC cells with EXO-miR-361-3p/EXO-miR-NC. B, F Relative expression of ETV7, BATF2, PAI-1 and p-ERK/T-ERK protein after ETV7 and/or BATF2 knockdown or overexpression (*P < 0.05, **P < 0.01, ***P < 0.001). CellTiter-Glo assays (C, G), wound healing assays (D, H) and Transwell assays (E, I) were performed to test the effect of ETV7 and/or BATF2 knockdown or overexpression on BC cell proliferation and metastasis. Scale bars, 100 μm

Fig. 4

MiR-361-3p promotes proliferation and migration by upregulating the PAI-1/ERK pathway in BC cells. A, B Relative expression of PAI-1 and p-ERK/T-ERK protein after transfection with PAI-1 plasmid alone or in combination with miR-361-3p and siPAI-1. CellTiter-Glo assays (C), Transwell assays (D), and wound healing assays (E) were performed to test the effect of overexpression of PAI-1 alone or in combination with miR-361-3p and knockdown of PAI-1 on BC cell proliferation and migration. Scale bars, 100 μm. F Analyzing the correlation between miR-361-3p and PAI-1 expression level using TCGA dataset. G GSEA analysis based on PAI-1 expression level using TCGA dataset. H, I Expression trend of PAI-1 in normal breast tissue, nonmetastatic BC tissue, and metastatic BC tissue. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Elevated plasma exosomal miR-361-3p promotes the malignant progression of BC in mice. A Upper panel: A schematic diagram of the animal experiments. Lower panel: A T47D cell xenograft model in female BALB/c nude mice was established. Mice exhibiting a high abundance of plasma exosomal miR-361-3p, as well as control mice, were obtained through the tail vein injection of EXO-miR-361-3p or EXO-miR-NC, respectively. B Differences in volume, weight and growth rate of murine tumors in the EXO-miR-361-3p group compared to the control group. C H&E staining of murine tumor tissues (×40). D–F Compare the expression levels of miR-361-3p, ETV7, BATF2, and the PAI-1/ERK pathway in excised tumors of the two groups of mice. G Proposed model of exosomal miR-361-3p targeting ETVT and BATF2 to upregulate the PAI-1/ERK pathway, leading to increased viability in BC cells. (*P < 0.05, **P < 0.01, ***P < 0.001)