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. 2021 Mar 26;13(7):9911-9926.
doi: 10.18632/aging.202747. Epub 2021 Mar 26.

Exosomes derived from microRNA-512-5p-transfected bone mesenchymal stem cells inhibit glioblastoma progression by targeting JAG1

Tengfeng Yan  1 , Miaojing Wu  1 , Shigang Lv  1   2 , Qing Hu  1 , Wenhua Xu  3 , Ailiang Zeng  2 , Kai Huang  1 , Xingen Zhu  1
Affiliations

Exosomes derived from microRNA-512-5p-transfected bone mesenchymal stem cells inhibit glioblastoma progression by targeting JAG1

Tengfeng Yan et al. Aging (Albany NY). .

Abstract

In this study, we demonstrate that bone mesenchymal stem cell (BMSC)-derived exosomes alter tumor phenotypes by delivering miR-512-5p. miR-512-5p was downregulated in glioblastoma tissues and cells, and Jagged 1 (JAG1) was the target gene of miR-512-5p. We clarified the expression patterns of miR-512-5p and JAG1 along with their interactions in glioblastoma. Additionally, we observed that BMSC-derived exosomes could contain and transport miR-512-5p to glioblastoma cells in vitro. BMSC-derived exosomal miR-512-5p inhibited glioblastoma cell proliferation and induced cell cycle arrest by suppressing JAG1 expression. In vivo assays validated the in vitro findings, with BMSC-exosomal miR-512-5p inhibiting glioblastoma growth and prolonging survival in mice. These results suggest that BMSC-derived exosomes transport miR-512-5p into glioblastoma and slow its progression by targeting JAG1. This study reveals a new molecular mechanism for glioblastoma treatment and validates miRNA packaging into exosomes for glioblastoma cell communication.

Keywords: bone mesenchymal stem cell (BMSC); exosomes; glioblastoma (GBM); jagged 1 (JAG1); miR-512-5p.

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Conflict of interest statement

CONFLICTS OF INTEREST: All authors declare no conflict of interest.

Figures

Figure 1
Figure 1
miR-512-5p is poorly expressed in GBM specimens and cell lines. (A) miR-512-5p expression in glioma tissues from the CCGA database as determined by RT-qPCR. (**p < 0.01, Student's t-test) (B) miR-512-5p expression in non-tumor brain tissues (NBTs) and GBM tissues as detected by FISH (200 ×). (C) miR-512-5p expression in five GBM and NHA cell lines as determined by RT-qPCR. (*p < 0.05, **p < 0.01, one-way ANOVA, compared NHA with GBM cell lines). Data are represented as the mean ± standard deviation of three independent experiments.
Figure 2
Figure 2
Overexpression of miR-512-5p inhibits GBM cell proliferation and causes cell cycle arrest. (A) miR-512-5p expression in U87 and LN229 cells determined after transfecting cells with corresponding vectors by RT-qPCR. (B) Cell proliferation activity as examined by EdU assays. (200 ×) (C) Cell proliferation activity as examined by colony formation assays. (D) Analysis of cell cycle by flow cytometry. (E) G1-arrest-relevant cell cycle regulators determined by western blotting. Data are represented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, Student's t-test, compared to the Control group.
Figure 3
Figure 3
miR-512-5p targets JAG1. (A) Venn diagrams show the targets of miR-512-5p. (B) The predicted miR-512-5p binding site on JAG1 3'-UTR (wild type, WT) is shown in red. (C) Dual luciferase reporter assays were used to confirm the binding site of miR-512-5p and JAG1. ( n.s, no significance, *p < 0.05, **p < 0.01, Student's t-test, compared to the JAG1-WT group.) (D) JAG1 expression in NBTs (n = 8) and glioma tissues (n = 71) as determined by western blotting. (*p < 0.05, Student's t-test) (E) The relationship between miR-512-5p and JAG1 was performed by Pearson’s correlation test (r = -0.3352, represent inverse relationship). (F) JAG1 expression determined after transfecting cells with corresponding vectors by western blotting analysis. *p < 0.05, **p < 0.01, Student's t-test, compared to the Control group. Data are represented as the mean ± standard deviation of three independent experiments.
Figure 4
Figure 4
JAG1 mediates the effect of miR-512-5p on GBM cells. (A) JAG1 expression determined by western blotting after transfecting U87-miR-512-5p or LN229-anti-miR-512-5p cells with JAG1 or sh-JAG1. (B) Cell proliferation activity as examined by EdU assays. (200 ×) (C) Cell proliferation activity as examined by colony formation assays. (D) Analysis of cell cycle by flow cytometry. (E) G1-arrest-relevant cell cycle regulators determined by western blotting. Data are represented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, Student's t-test, compared to the Control group.
Figure 5
Figure 5
BMSC-exosomal miR-512-5p can be internalized by U87 cells. (A) Surface marker proteins of BMSC identified by flow cytometry assays. (B) Representative TEM image of BMSC exosomes. (scale bar, 200 μM) (C) Particle size distribution of BMSC exosomes as measured by DLS. (D) Exosome surface markers determined by western blotting. (E) miR-512-5p expression found in BMSC and BMSC-derived exosomes by RT-qPCR after transfection with miR-512-5p or the Control. (F) BMSC-exosomal miR-512-5p internalized by U87 cells. (scar bar = 25 μM). (G) miR-512-5p and JAG1 expression determined by RT-qPCR in U87 cells after co-culture with BMSC-derived exosomes or PBS. (H) JAG1 expression determined by western blotting in U87 cells after co-culture with BMSC-derived exosomes or PBS. Data are represented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, Student's t-test, compared to the Control group.
Figure 6
Figure 6
BMSC-exosomal miR-512-5p inhibits GBM cell proliferation and causes cell cycle arrest. (A) Cell proliferation activity as examined by EdU assays. (200 ×) (B) Cell proliferation activity as examined by colony formation assays. (C) Analysis of cell cycle by flow cytometry. (D) G1-arrest-relevant cell cycle regulators determined by western blotting assays. Data are represented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, Student's t-test, compared to the Control group.
Figure 7
Figure 7
BMSC-exosomal miR-512-5p inhibits tumor growth in vivo. (A) Effect of BMSC-exosomal miR-512-5p as determined by HE in nude mice. (B) JAG1 and Ki-67 expression as determined by IHC. (scale bar = 50 μM) (C) Tumor growth as determined by bioluminescence imaging. after treatment with PBS or BMSC-derived exosomes. (D) Overall survival of nude mice receiving treatment was determined by Kaplan-Meier analysis. (n = 15, respectively). (*p = 0.0184, comparing the BMSC-Control-Exo group with the PBS group, #p = 0.0036, comparing the BMSC-miR-512-5p-Exo group with the PBS and BMSC-Control-Exo groups, Log-Rank test).
Figure 8
Figure 8
Exosomes derived from MicroRNA-512-5p transfected bone mesenchymal stem cell inhibit glioblastoma progression by targeting JAG1. BMSC-exosomal miR-512-5p inhibited the expression of JAG1 in GBM, and suppressed cell proliferation and G1-S phase cell cycle by inhibiting the expression of CDK4, CDK6 and Cyclin D1.
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