Inhibition of glioblastoma cell invasion by hsa-miR-145-5p and hsa-miR-31-5p co-overexpression in human mesenchymal stem cells

Restricted access

OBJECTIVE

Human bone marrow–derived mesenchymal stem cells (hMSCs) show tropism for brain tumors and may be a useful vehicle for drug or gene delivery to malignant gliomas. Recently, some microRNAs (miRNAs) have been shown to suppress the invasiveness of malignant gliomas.

METHODS

To test their potential to become vehicles for the delivery of miRNA to malignant gliomas, hMSCs were engineered so that hMSC secretion of miRNAs that inhibit glioma cell invasion was enabled without altering the hMSC tropism for glioma cells.

RESULTS

In coculture, hMSCs cotransfected with hsa-miR-145-5p and -31-5p miRNAs showed markedly reduced invasion by U87 glioma cells in a contact-dependent manner both in vitro and ex vivo, with invasion of hMSCs cotransfected with these 2 miRNAs by the U87 cells reduced to 60.7% compared with control cells. According to a Matrigel invasion assay, the tropism of the hMSCs for U87 cells was not affected. In glioma cell lines U251 and LN229, hMSCs exhibited tropism in vivo, and invasion of hMSCs cotransfected with hsa-miR-145-5p and -31-5p was also significantly less than that of control cells. When U87 cells were coimplanted into the striatum of organotypic rat brain slices with hMSCs cotransfected with hsa-miR-145 and -31-5p, the relative invasive area decreased by 37.1%; interestingly, these U87 cells showed a change to a rounded morphology that was apparent at the invasion front. Whole-genome microarray analysis of the expression levels of 58,341 genes revealed that the co-overexpression of hsa-miR-145-5p and -31-5p downregulated FSCN1 expression in U87 cells.

CONCLUSIONS

This study demonstrates that miRNA overexpression in hMSCs can alter the function of glioma cells via contact-dependent transfer. Co-overexpression of multiple miRNAs may be a useful and novel therapeutic strategy. The study results suggest that hMSCs can be applied as a delivery vehicle for miRNAs.

ABBREVIATIONS α-MEM = alpha minimum essential medium; cDNA = complementary DNA; Cq = quantitation cycle; FBS = fetal bovine serum; FSCN1 = fascin actin-bundling protein 1; GBM = glioblastoma; GFP = green fluorescent protein; hMSC = human bone marrow–derived mesenchymal stem cell; miRNA = microRNA; PBS = phosphate-buffered saline; PCR = polymerase chain reaction.

Downloadable materials

  • Supplementary Figures and Tables (PDF 1.09 MB)

Article Information

Correspondence Akira Nakamizo: Kyushu Medical Center, Fukuoka, Japan. nakamizo@ns.med.kyushu-u.ac.jp.

INCLUDE WHEN CITING Published online March 9, 2018; DOI: 10.3171/2017.8.JNS1788.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    A–D: Results of cell invasion analysis using the Matrigel invasion assay. Negative control (NC) was used at 10, 20, and 30 nM for miRNA alone, combinations of 2, or combinations of 3 miRNAs, respectively (black bars). Data are shown as the means ± SEM (whiskers) (n = 3). *p < 0.05. A: Effects of miRNAs on the invasive ability of U87 cells. U87 cells overexpressing miRNA were placed in the upper wells of Transwell plates, and α-MEM supplemented with 10% FBS was placed in the lower wells. B: Effect of miRNAs on the tropism of hMSCs for U87 cells. hMSCs overexpressing miRNA were placed in the upper wells of Transwell plates, and conditioned medium obtained from U87 cells was placed in the lower wells. C: Effect of coculturing hMSCs transfected with miRNAs on the invasive ability of U87 cells via contact-dependent transfer. U87 cells and miRNA-overexpressing hMSCs were placed at a 1:1 ratio in the upper wells of Transwell plates, and α-MEM supplemented with 10% FBS was placed in the lower wells. D: Effect of the conditioned medium obtained from hMSCs transfected with miRNAs on the invasion of U87 cells. U87 cells were placed in the upper wells of the Transwell plates and incubated with conditioned medium obtained from hMSCs transfected with miRNA mimics, and α-MEM supplemented with 10% FBS was placed in the lower wells. E: Representative micrographs of the Matrigel invasion assay showing that coculturing hMSCs transfected with hsa-miR-145-5p and -31-5p significantly inhibited the invasion of U87 cells (upper) compared with coculturing hMSCs transfected with the 20 nM NC (lower). Bar = 200 mm. Figure is available in color online only.

  • View in gallery

    MicroRNA transfer from hMSCs to U87 cells in a contact-dependent manner. Human bone marrow–derived MSCs transfected with Alexa555-labeled hsa-miR-31-5p were cocultured with U87 cells. Twenty-four hours after coculture, the cells were analyzed using fluorescence microscopy. A: Red shows hsa-miR-31-5p labeled with Alexa555. B: Green shows a U87 cell labeled with Vybrant DiO. C: The Alexa555-labeled hsa-miR-31-5p signal is localized in the U87 cell (arrow). Bar = 20 µm. Figure is available in color online only.

  • View in gallery

    Representative photomicrographs of cell aggregates containing U87 cells and hMSCs implanted into Sprague Dawley rat organotypic brain slices. GFP-expressing U87 cells were coimplanted with Vybrant DiI–labeled hMSCs transfected with hsa-miR-145-5p and -31-5p or the negative control into the striatum of the brain slice. The invasion of U87 cells cotransplanted with hMSCs that were cotransfected with hsa-miR-145-5p and -31-5p was significantly inhibited (lower) compared with the invasion of U87 cells cotransplanted with hMSCs that were transfected with the negative control (upper) at 4 days after implantation. High-magnification (×200) photomicrographs show a mainly spindle-shaped morphology (upper) for the invasion front of the U87 cells coimplanted with hMSCs that were transfected with the negative control and a mainly rounded morphology (lower) for U87 cells coimplanted with hMSCs that were cotransfected with hsa-miR-145-5p and -31-5p. Bar = 200 µm. Figure is available in color online only.

  • View in gallery

    A: Effect of hsa-miR-145-5p and -31-5p on the invasive ability of U87 cells. The relative invasive area of U87 cells coimplanted with hMSCs that were cotransfected with hsa-miR-145-5p and -31-5p (n = 6) was decreased by 37.1% compared with that of the U87 cells coimplanted with hMSCs that were transfected with the negative control (n = 6) on day 4. The area of the cells on the day of implantation was assigned a value of 100%. B: Effect of hsa-miR-145-5p and -31-5p on the tropism of hMSCs. We observed no significant difference in the migration area of the hMSCs cotransfected with hsa-miR-145-5p and -31-5p compared with the area of hMSCs transfected with the negative control on day 4. C: Effect of hsa-miR-145-5p and -31-5p on U87 cell proliferation. Cell proliferation in the cell aggregates was assessed by counting the number of U87 cells and hMSCs transfected with miRNAs or the negative control. We found no significant difference in the total number of cells between cell aggregates containing U87 cells and hMSCs transfected with the NC and aggregates containing U87 cells and hMSCs cotransfected with hsa-miR-145-5p and -31-5p at 4 days after seeding. Data are shown as the means ± SEM (error bars) (n = 6). *p < 0.05. n.s. = not significant.

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 117 117 56
Full Text Views 96 96 39
PDF Downloads 71 71 26
EPUB Downloads 0 0 0

PubMed

Google Scholar