Radiation-induced exosomal miR-21 enhances tumor proliferation and invasiveness in breast cancer: implications for poor prognosis in radiotherapy patients

Abstract

Radiotherapy is widely used as an effective non-surgical strategy to control malignant tumors. However, recurrence is one of common causes of treatment failure even after the effective radiotherapy. In this study, we focused on the effects of radiation-induced exosomal miR-21 on the tumor microenvironment to investigate the causes of recurrence. Analysis of the TCGA database revealed that breast cancer patients with high levels of miR-21 have significantly reduced overall survival when treated with radiotherapy compared to those who did not receive radiotherapy, indicating a high hazard ratio for miR-21 in patients undergoing this treatment. Additionally, exosomal miR-21 is found to be highly expressed in the serum of breast adenocarcinoma patients. To explore how miR-21 induces poor prognosis in irradiated breast cancer, we irradiated 4T1 cell line with low or high doses of radiation, and examined the impact of secreted exosomal miR-21 on breast cancer cell and tumor microenvironment. After 10 Gy irradiation, 4T1 cells secreted 2.20 ± 0.10 times more exosomes and exhibited a 1.85 ± 0.01-fold increase in exosomal miR-21 levels. Treatment with exosomes from 10 Gy-irradiated cancer cells led to enhanced tumor cell proliferation, wound healing, and migration. The survival rate of 10 Gy-irradiated tumor cells incubated with 10 Gy-derived exosomes increased by 2.83-fold. Moreover, the growth of subcutaneous tumors treated with 10 Gy exosomes (n = 13) was significantly faster compared to tumors treated with 0 Gy exosomes (n = 10, P < 0.05). In summary, our study revealed high-dose irradiation-induced exosomes were found to enhance tumor proliferation and invasiveness via the transfer of exosomal miR-21. Based on these findings, we suggest that radiation-induced exosomal miR-21 may contribute to a poorer prognosis of breast cancer patients undergoing radiotherapy.

Keywords: Exosomal miR-21; Poor prognosis of breast cancer patients; Radiation-induced exosomes; Radiotherapy.

Fig. 1

Breast cancer patients with high miR-21 levels are correlated with poor prognosis after radiation therapy. (A) Kaplan-Meier survival analysis of the TCGA breast cancer cohort stratified by radiotherapy status and miR-21 expression levels. Sample sizes are indicated at specific time points. (B) Hazard ratios for groups stratified by radiotherapy status and miR-21 expression levels. Cox proportional hazards model was used to calculate hazard ratios and corresponding p-values for each group. (C) Expression of has-miR-21 in breast cancer patients stratified by RX status. (D) Pearson correlation analysis between miR-21 expression and overall survival for each sample. (E, F) Representative images (E) and survival fraction (graph, F) of Clonogenic assay in 4T1 after irradiation (0/2/5/10 Gy). (G) Cell viability test in 4T1 at 24 and 48 h after irradiation (0/2/5/10 Gy). (H) miR-21 expression in 4T1 after irradiation (0/5/10 Gy). (I) Comparative analysis of exosomal miR-21 expression (RPM, reads per million) in breast adenocarcinoma and healthy control. (J) Distribution of exosomal miRNA expression in breast adenocarcinoma samples from the EVmiRNA database. The levels of hsa-miR-21-5p were compared with the distribution of other miRNAs available in the EVmiR database. Empirical p-value was determined based on the rank of hsa-miR-21-5p expression within the expression ranks of other miRNAs. (K) Experimental scheme for isolating exosomes. (L, M) The amount of secreted exosomes according to irradiation dose (0/5/10/25/50 Gy, L) and that normalized with viable cell number (M). (N) Quantification of exosomal miR-21 levels isolated from irradiated 4T1 (0/5/10 Gy)

Fig. 2

(A) Vector construct. (B) Bioluminescence imaging strategy for the miR-21 reporter system. (C) Fluorescence images of 4T1 with Alexa 488-labeled exosomes. (D, E) Bioluminescence images (D) and luciferase assay (E) of 4T1/miR-21-luc treated with exosomes (0/5/10 Gy). (F) Cell proliferation of 4T1 with exosomes (0/5/10 Gy). (G) Survival fraction of irradiated 4T1 with exosomes (0/5/10 Gy). (H) Relative survival value in 10 Gy irradiated 4T1 treated with exosomes (0/5/10 Gy). (I) Xenograft tumor volume with exosome (0/5/10 Gy) treatment. (J, K) Representative images of migration assay of 4T1 with exosomes (J) and migration ratio (graph, K). (L, M) Representative images of wound healing assay (L) and distance analysis (graph, M) of 4T1 with exosomes (0/5/10 Gy)