Exosome-transmitted microRNA-133b inhibited bladder cancer proliferation by upregulating dual-specificity protein phosphatase 1

Abstract

Bladder Cancer (BC) is the ninth most common tumor in the world and one of the most common malignant tumors of the urinary system. Some studies reported that miR-133b expression is reduced in BC, but whether it plays a role in the development of BC and its mechanism is unclear. microRNAs can be packaged into exosomes to mediate communication between tumor cells, affecting their proliferation and apoptosis. The objective of this study was to investigate the effect of exosomal miR-133b on BC proliferation and its molecular mechanism. Firstly, the expression of miR-133b was evaluated in BC and adjacent normal tissues, as well as in serum exosomes of BC patients and healthy controls. Then the delivery and internalization of exosomes in cells was observed through fluorescence localization. Cell viability and apoptosis were assessed in BC cells transfected with mimics and incubated with exosomes. The role of exosomal miR-133b was also analyzed in nude mice transplant tumors. Furthermore, the target gene of miR-133b was predicted through bioinformatics. The level of miR-133b was significantly decreased in BC tissues and in exosomes from serum of patients, which was correlated with poor overall survival in TCGA. Exosomal miR-133b could be obtained using BC cells after transfection with miR-133b mimics. The miR-133b expression increased after incubation with exosomal miR-133b, which lead to the inhibition of viability and increase of apoptosis in BC cells. Exosomal miR-133b could suppress tumor growth in vivo. In addition, we found that exosomal miR-133b may play a role in suppressing BC proliferation by upregulating dual-specificity protein phosphatase 1 (DUSP1). These findings may offer promise for new therapeutic directions of BC.

Keywords: Bladder cancer; Dual-specificity protein phosphatase 1; Exosome; Proliferation; microRNA-133b.

Figure 1

miR‐133b expression was significantly downregulated in BC tissues, and was correlated with poor overall survival in TCGA. Relative expressions of miR‐133b in BC tissues and adjacent normal tissues (A). BC patients with low miR‐133b expression had lower overall survival rates than patients with high miR‐133b expression in the TCGA cohort (B) (P < .001). *P < .05

Figure 2

Expression of serum exosomal miR‐133b in patients with bladder cancer. Transmission electron microscopy image of exosomes derived from the serum of patients and controls. Scale bars represent 100 nm (A). Western blotting analysis showing the presence of CD63 and CD81 in exosomes (B). The expression of miR‐133b was detected in serum and serum exosomes (C). The relative expression levels of exosomal miR‐133b were stable after storing at −80°C, 4°C and room temperature for 12 hours respectively (D). qRT‐PCR detection of miR‐133b in exosomes from serum (E). *P < .05

Figure 3

Effect of miR‐133b on bladder cancer cellular phenotype. 5637 and T24 cells were transfected with NC or miR‐133b mimics. The expression of miR‐133b in 5637 and T24 cells (A). A CCK8 assay detection of cell viability (B). Colony formation assays for evaluation of cell proliferation (C). Flow cytometry detection of the apoptosis of 5637 and T24 cells (D). *P < .05

Figure 4

Exosomal miR‐133b act as a mediator for intercellular communication. The expression of miR‐133b in the medium of 5637 and T24 cells treated with RNase (3 μg/ml) alone or combined with Triton X‐100 (0.3%) for 20 minutes (A). Transmission electron microscopy image of exosomes derived from the serum of patients and controls. Scale bars represent 100 nm (B). Western blotting analysis showing the presence of CD63 and CD81 in exosomes (C). Confocal microscopy image showing the internalization of PKH67‐labeled exosomes derived from 5637 and T24 cells for three hours, green represents PKH67, and blue represents nuclear DNA staining by DAPI (D). *P < .05

Figure 5

Effect of exosomal miR‐133b on bladder cancer cellular phenotype. Exosomes were isolated from 5637 and T24 cells transfected with NC or miR‐133b mimics, namely NC‐EXO and miR‐133b‐EXO, respectively. Their exosomes were extracted and cocultured with BC cells for 24 hours. A CCK8 assay detection of cell viability (A). The expression of miR‐133b in 5637 and T24 cells (B). Colony formation assays for evaluation of cell proliferation (C). Flow cytometry detection of the apoptosis of 5637 and T24 cells (D). *P < .05

Figure 6

DUSP1 is a direct target of miR‐133b. The two target sites for miR‐133b within the DUSP1 promoter RNA (A). The expression of DUSP1 mRNA in BC tissues and adjacent normal tissues (B). miR‐133b upregulated the expression of DUSP1 in 5637 and T24 cells via transfection with miR‐133b mimics (C). DUSP1 mRNA was upregulated in 5637 and T24 cells after incubated with miR‐133b‐EXO (D). Western blots of DUSP1 in T24 cells were treated with NC or miR‐133b mimics and NC‐EXO or miR‐133b‐EXO (E). *P < .05

Figure 7

Effect of miR‐133b overexpression and exosomal miR‐133b on tumor in vivo. Xenograft model in nude mice. Red arrows show position of tumor (A). The excision tumors of T24 xenografts inoculated in agomir‐NC, agomir‐miR‐133b, NC‐EXO, miR‐133b‐EXO (B). The tumor volumes and weights of the agormir‐miR‐133b and miR‐133b‐EXO groups were significantly reduced compared to their counterparts in the control groups (C‐D). Detection of miR‐133b and DUSP1 mRNA expression in tumor tissues of nude mice treated with agomir‐NC, agomir‐miR‐133b, NC‐EXO, miR‐133b‐EXO by qRT‐PCR (E‐F). Western blots showed the expression of DUSP1 proteins in tumor tissues (G). *P < .05

Figure 8

Schematic diagram of exosomal miR‐133b‐mediated BC proliferation. Exosomal miR‐133b derived from BC cells transfected with miR‐133b mimics, and then obtained from other BC cells again, to suppress the proliferation of BC by targeting DUSP1