Mesenchymal stem cell-derived exosomal microRNA-133b suppresses glioma progression via Wnt/β-catenin signaling pathway by targeting EZH2

Abstract

Background: Mesenchymal stem cells (MSCs) play a significant role in cancer initiation and metastasis, sometimes by releasing exosomes that mediate cell communication by delivering microRNAs (miRNAs). This study aimed to investigate the effects of exosomal miR-133b derived from MSCs on glioma cell behaviors.

Methods: Microarray-based analysis identified the differentially expressed genes (DEGs) in glioma. The expression patterns of EZH2 and miR-133b along with interaction between them were clarified in glioma. The expression of miR-133b and EZH2 in glioma cells was altered to examine their functions on cell activities. Furthermore, glioma cells were co-cultured with MSC-derived exosomes treated with miR-133b mimic or inhibitor, and EZH2-over-expressing vectors or shRNA against EZH2 to characterize their effect on proliferation, invasion, and migration of glioma cells in vitro. In vivo assays were also performed to validate the in vitro findings.

Results: miR-133b was downregulated while EZH2 was upregulated in glioma tissues and cells. miR-133b was found to target and negatively regulate EZH2 expression. Moreover, EZH2 silencing resulted in inhibited glioma cell proliferation, invasion, and migration. Additionally, MSC-derived exosomes containing miR-133b repressed glioma cell proliferation, invasion, and migration by inhibiting EZH2 and the Wnt/β-catenin signaling pathway. Furthermore, in vivo experiments confirmed the tumor-suppressive effects of MSC-derived exosomal miR-133b on glioma development.

Conclusion: Collectively, the obtained results suggested that MSC-derived exosomes carrying miR-133b could attenuate glioma development via disrupting the Wnt/β-catenin signaling pathway by inhibiting EZH2, which provides a potential treatment biomarker for glioma.

Keywords: EZH2; Exosomes; Glioma; Mesenchymal stem cells; MicroRNA-133b; Wnt/β-catenin signaling pathway.

Fig. 1

EZH2 is highly expressed in glioma tissues and cells. a The intersection of the top 700 DEGs in GSE12657, GSE35493, and GSE50161. b, c Heat maps of DEGs in GSE35493 and GSE50161. d EZH2 gene expression profile analysis in GSE12657; green represents the gene expression of normal samples, and red represents the gene expression of glioma samples. e EZH2 gene expression determined in normal and glioma samples based on the TCGA database. f Gene network map of EZH2 and its related genes generated using Cytospace software. g EZH2 mRNA level measured in normal brain tissues (n = 12) and glioma tissues (n = 54) using RT-qPCR. h EZH2 protein level measured in normal brain tissues (n = 12) and glioma tissues (n = 54) using Western blot analysis. i Positive expression of EZH2 detected in glioma using immunohistochemistry (200×). j EZH2 expression measured in human normal brain glial cell line HEB and human glioma cell lines (U87, U251, LN229, and A172) using RT-qPCR; *p < 0.05 vs. normal brain tissues/HEB cell line; measurement data were shown as mean ± standard error; comparisons between two groups were conducted by unpaired t test, and comparisons among multiple groups were assessed by one-way analysis of variance (Dunnett’s post hoc test). The experiment was repeated three times to obtain the mean value

Fig. 2

Silencing of EZH2 suppresses cell proliferation, invasion, and migration in glioma. a EZH2 mRNA level determined in glioma cells treated with oe-EZH2 or sh-EZH2 using RT-qPCR. b EZH2 protein band patterns detected in glioma cells treated with oe-EZH2 or sh-EZH2. c EZH2 protein level measured in glioma cells treated with oe-EZH2 or sh-EZH2 using Western blot analysis. d Proliferation of glioma cells treated with oe-EZH2 or sh-EZH2 detected using EdU assay (200×). e Migration of glioma cells treated with oe-EZH2 or sh-EZH2 detected using Transwell assay (200×). f Invasion of glioma cells treated with oe-EZH2 or sh-EZH2 detected using Transwell assay (200×); ^#p < 0.05 vs. glioma cells treated with oe-NC; ^&p < 0.05 vs. glioma cells treated with sh-NC; measurement data were shown as mean ± standard error; comparisons between two groups were conducted by unpaired t test. The experiment was repeated three times to obtain the mean value

Fig. 3

miR-133b targets and negatively regulates EZH2. a Venn analysis of miRNAs that regulate EZH2; blue represents the predicted results of DIANA database, red represents the predicted results of mirDIP database, green represents the predicted results of mirWalk database, and yellow represents the differentially expressed downregulated miRNAs in GSE42658. b miR-133b expression in normal brain tissues (n = 12) and glioma tissues (n = 54) determined by RT-qPCR. c Binding site prediction of miR-133b in EZH2 3′UTR. d Detection of luciferase activity using dual-luciferase reporter gene assay. e miR-133b expression and EZH2 mRNA level after miR-133b over-expression and inhibition using RT-qPCR. f EZH2 protein level after miR-133b over-expression and inhibition using Western blot analysis; *p < 0.05 vs. normal brain tissues; ^#p < 0.05 vs. glioma cells treated with mimic-NC; ^&p < 0.05 vs. glioma cells treated with inhibitor-NC; measurement data were shown as mean ± standard deviation; comparisons between two groups were conducted by unpaired t test, and comparisons among multiple groups were assessed by one-way analysis of variance (Dunnett’s post hoc test). The experiment was repeated three times to obtain the mean value

Fig. 4

Characterization of MSCs and MSC-secreted exosomes. a Morphological observation of MSCs (100×). b Identification of surface marker molecules in MSCs using flow cytometry. c Osteogenic and adipogenic induction culture of MSCs (400×). (a) Osteogenic differentiation evaluated by alizarin red staining; (b) adipogenic differentiation evaluated by oil red O staining; (c) chondral differentiation-Alcian blue staining (200×). d Identification of exosomes by a transmission electron microscopy. e Detection of exosome diameter by dynamic light scattering. f Exosome surface markers CD63 and HSP70 measured using Western blot analysis; lane 1, extracted exosomes; lane 2, supernatant after extraction of exosomes. g The content of CD63, a surface marker in exosomes, detected using flow cytometry; *p < 0.05 vs. lane 1; measurement data were shown as mean ± standard error. The experiment was repeated three times to obtain the mean value

Fig. 5

MSCs transfer miR-133b to glioma cells through exosomes to repress the expression of EZH2. a Expression of miR-133b and EZH2 in MSCs co-cultured with glioma cells after restoration or depletion of miR-133b determine using RT-qPCR. b Internalization of exosomes by glioma cells observed under the inverted microscope (400×). c miR-133b and EZH2 expression in MSCs treated with GW4869 co-cultured glioma cells after restoration or depletion of miR-133b measured using RT-qPCR. d miR-133b expression in exosomes and MSCs after restoration or depletion of miR-133b measured using RT-qPCR. e miR-133b expression in glioma cells co-cultured with MSC-derived exosome by RT-qPCR; ^#p < 0.05 vs. MSCs co-cultured with glioma cells treated with mimic-NC; ^&p < 0.05 vs. MSCs co-cultured with glioma cells treated with inhibitor-NC; *p < 0.05 vs. glioma cells co-cultured with PBS; ^{^}p < 0.05 vs. glioma cells co-cultured with miR-133b mimic NC; ^@p < 0.05 vs. glioma cells co-cultured with miR-133b inhibitor NC; measurement data were shown as mean ± standard deviation; comparisons among multiple groups were assessed by one-way analysis of variance (Dunnett’s post hoc test). The experiment was repeated three times to obtain the mean value

Fig. 6

Exosomal miR-133b derived from MSCs represses glioma cell proliferation, migration, and invasion via inhibition of the Wnt/β-catenin signaling pathway. a Proliferation of glioma cells treated with exo-miR-133b mimic, exo-miR-133b inhibitor, or oe-EZH2 + exo-miR-133b mimic detected using EdU assay (200×). b Migration of glioma cells treated with exo-miR-133b mimic, exo-miR-133b inhibitor, or oe-EZH2 + exo-miR-133b mimic detected using Transwell assay (200×). c Invasion of glioma cells treated with exo-miR-133b mimic, exo-miR-133b inhibitor, or oe-EZH2 + exo-miR-133b mimic detected using Transwell assay (200×). d Protein band patterns of EZH2, Wnt1, p-GSK-3β, GSK-3β, and β-catenin detected in glioma cells treated with exo-miR-133b mimic, exo-miR-133b inhibitor, or oe-EZH2 + exo-miR-133b mimic. e Protein levels of EZH2, Wnt1, p-GSK-3β, GSK-3β, and β-catenin measured in glioma cells treated with exo-miR-133b mimic, exo-miR-133b inhibitor, or oe-EZH2 + exo-miR-133b mimic using Western blot analysis; ^#p < 0.05 vs. glioma cells treated with exo-mimic NC; ^&p < 0.05 vs. glioma cells treated with exo-inhibitor NC; measurement data were shown as mean ± standard deviation; comparisons among multiple groups were assessed by one-way analysis of variance (Dunnett’s post hoc test). The experiment was repeated three times to obtain the mean value

Fig. 7

MSC-derived exosomal miR-133b suppressed tumor growth of glioma in vivo. a Tumorigenesis of nude mice (n = 6). b Tumor volume of nude mice (n = 6). c Tumor weight of nude mice (n = 6). d Expression of miR-133b and EZH2 determined using RT-qPCR. e Protein levels of EZH2, Wnt1, p-GSK-3β, GSK-3β, and β-catenin measured using Western blot analysis; ^#p < 0.05 vs. nude mice injected with exo-mimic NC; ^&p < 0.05 vs. glioma cells injected with exo-inhibitor NC; measurement data were shown as mean ± standard deviation; comparisons between two groups were conducted by unpaired t test; The data analysis at different time points was performed by repeated measures ANOVA with Bonferroni post hoc test. The experiment was repeated three times to obtain the mean value

Fig. 8

The schematic representation of mechanism by which MSC-derived exosomes containing miR-133b affect glioma cell activities. MSCs transfer miR-133b to glioma cells through exosomes to inhibit the EZH2 expression via suppression of the Wnt/β-catenin signaling pathway, whereby the glioma cell proliferation, migration, and invasion are diminished, and ultimately, the progression of glioma is attenuated