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. 2020 Apr 29;6(18):eaay3051.
doi: 10.1126/sciadv.aay3051. eCollection 2020 May.

miR-26a regulates extracellular vesicle secretion from prostate cancer cells via targeting SHC4, PFDN4, and CHORDC1

Fumihiko Urabe  1   2   3 , Nobuyoshi Kosaka  1 , Yurika Sawa  1 , Yusuke Yamamoto  3 , Kagenori Ito  2   3 , Tomofumi Yamamoto  1 , Takahiro Kimura  2 , Shin Egawa  2 , Takahiro Ochiya  1   3
Affiliations

miR-26a regulates extracellular vesicle secretion from prostate cancer cells via targeting SHC4, PFDN4, and CHORDC1

Fumihiko Urabe et al. Sci Adv. .

Abstract

Extracellular vesicles (EVs) are involved in intercellular communication during cancer progression; thus, elucidating the mechanism of EV secretion in cancer cells will contribute to the development of an EV-targeted cancer treatment. However, the biogenesis of EVs in cancer cells is not fully understood. MicroRNAs (miRNAs) regulate a variety of biological phenomena; thus, miRNAs could regulate EV secretion. Here, we performed high-throughput miRNA-based screening to identify the regulators of EV secretion using an ExoScreen assay. By using this method, we identified miR-26a involved in EV secretion from prostate cancer (PCa) cells. In addition, we found that SHC4, PFDN4, and CHORDC1 genes regulate EV secretion in PCa cells. Furthermore, the progression of the PCa cells suppressing these genes was inhibited in an in vivo study. Together, our findings suggest that miR-26a regulates EV secretion via targeting SHC4, PFDN4, and CHORDC1 in PCa cells, resulting in the suppression of PCa progression.

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Figures

Fig. 1
Fig. 1. Screening of miRNAs regulating EV secretion in PCa.
(A) Schematic illustration of a high-throughput compatible EV biogenesis assay to detect EV biogenesis–regulating miRNAs. (B) Immunoblot analysis of the conventional EV markers. A 1-μg sample of EVs and a 10-μg sample of cell lysate from PC3M cells were loaded into each lane. (C) Flow diagram of miRNAs used for selecting candidate miRNAs. (D) Venn diagram showing miRNAs that suppress EV secretion. The miRNAs whose relative EV secretion/cell viability was lower than 0.8 were selected in each assay. The secretion of EVs was evaluated via ExoScreen, and the cell viability was measured via MTS assay. (E) Expression levels of miR-26a and miR-194 in PCa clinical specimens (GSE21036). **P < 0.01; and n.s., not significant. (F) Effect of the miR-26a mimic on EV secretion per PC3M cell. The amount of secretion of EVs per cell was evaluated by the signal intensity of ExoScreen per cell. The values are depicted as the fold change relative to the nonspecific miRNA mimic (control). The values are the means ± SE (n = 3). **P < 0.01. (G) Effect of the miR-26a mimic on EV secretion per PC3M cell. The amount of EV secreted per cell was evaluated using a nanoparticle tracking system. The values are the means ± SE (n = 3). **P < 0.01.
Fig. 2
Fig. 2. SHC4, PFDN4, and CHORDC1 are involved in miR-26a–mediated EV secretion.
(A) Venn diagram of predicted miR-26a targets (TargetScan) and transcripts that were experimentally repressed >2-fold by miR-26a overexpression in PCa cells (PC3M or PC3) relative to control conditions. (B) Schematic of the high-throughput compatible EV biogenesis assay to choose EV biogenesis–regulating genes. (C) Venn diagram showing genes that suppress EV secretion evaluated by ExoScreen. The genes whose relative EV secretion/cell viability was lower than that of miR-26a plus 0.3 were selected in each assay. The secretion of EV was evaluated by ExoScreen, and the cell viability was measured through the MTS assay. (D) Effect of siRNAs against candidate genes on EV secretion in PC3M cells. The EV secretion per cell was evaluated by the signal intensity of ExoScreen per cell. The values are depicted as the fold change relative to the negative control siRNA (control). The values are the means ± SE (n = 3). *P < 0.05; **P < 0.01; and n.s., not significant. (E) Effect of siRNAs against candidate genes on EV secretion per PC3M cell. The particle number of EVs was measured using a nanoparticle tracking system. The values are the means ± SE (n = 3). *P < 0.05; n.s., not significant. (F) Effect of SHC4, PFDN4, and CHORDC1 siRNA on the mRNA expression level of each gene. β-Actin was used as an internal control. Error bars represent the SE deduced by Student’s t test (*P < 0.05 and **P < 0.01). n.s., no significant difference. The data are representative of at least three independent experiments. The values are the means ± SE (n = 3). **P < 0.01.
Fig. 3
Fig. 3. miR-26a directly regulates the expression levels of SHC4, PFDN4, and CHORDC1.
(A) Immunoblot analysis of PC3M cells transfected with nonspecific miRNA mimic (negative control mimic) or miR-26a mimic. A 25-μg sample of cell lysate derived from transfected PCa cells was loaded for the detection of SHC4, CHORDC1, PFDN4, and actin. (B) Summary of miR-26a target sites and mutated sites (shown in red) in the 3′UTRs of SHC4, PFDN4, and CHORDC1. (C) Target validation of SHC4, PFDN4, and CHORDC1 was confirmed in the luciferase reporter assay. The values are depicted as the fold change relative to the negative control siRNA (control). wt, wild type; mut, mutant. The values are the means ± SE (n = 8, ×3 independent experiments). **P < 0.01. (D) Expression levels of SHC4, PFDN4, and CHORDC1 in PCa and normal prostate tissue clinical specimens (GSE6099). *P < 0.05; and n.s., not significant.
Fig. 4
Fig. 4. Down-regulation of EV secretion inhibits cancer progression in vivo.
(A) Establishing the PC3M cell line with stable SHC4, PFDN4, and CHORDC1 depletion using shRNAs and evaluation of EV secretion. The values are the means ± SE (n = 3). **P < 0.01. (B) The tumor volumes were measured every 3 days after tumor inoculation. The values are the means ± SE (n = 3). *P < 0.05; **P < 0.01. (C) The tumor weights in nude mice at day 21 were determined. The values are the means ± SE (n = 3). *P < 0.05; **P < 0.01. (D) The tumor volumes were measured every other day before the injection of EVs. The values are the means ± SE (n = 6).
Fig. 5
Fig. 5. Schematic model of the regulation of EV secretion in PCa.
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