Exosomal excretion of miR-3 7 5 promotes glioma progression by activating the CTGF-EGF R  pathway


 Background: Exosomes are membrane-bound extracellular vesicles of 40–150 nm produced by many cell types, and playing an important role in the maintenance of cellular homeostasis. Exosome secretion allows for the selective removal of harmful substances from cells. Whether this process also takes place in glioma cells is unknown. Methods: The role of the tumour suppressor miR-375 was explored in human glioma cell lines. Immunoblotting and qRT-PCR experiments demonstrated a functional link between miR-375 and its target, connective tissue growth factor (CTGF), and led to identify the involved molecular pathways. The exosomes secreted by glioma cells were extracted by ultracentrifugation and examined by transmission electron microscopy. The exosomal expression of miR-375 was analyzed by qRT-PCR. The exosome secretion inhibitor, GW4869, was used to study the biological significance of miR-375 release. Moreover, the dynamics of miR-375 release by glioma cells was investigated by using fluorescently labelled exosomes. Finally, exosomal miR-375 release was studied in an orthotopic xenograft model in nude mice. Results: MiR-375 expression was downregulated in gliomas. MiR-375 suppressed glioma proliferation, migration, and invasion by inhibiting the CTGF-epidermal growth factor receptor (EGFR) signalling pathway. MiR-375-containing exosomes were also found in human peripheral blood samples from glioma patients, and their level was correlated with the status of disease progression. Exosomal miR-375 secretion had an impact on the activity of the CTGF-EGFR pathway. Once secreted, exosomal miR-375 was not reuptaken by glioma cells. Conclusions: Exosomal miR-375 secretion allowed for sustained activation of the CTGF-EGFR oncogenic pathway, promoting the proliferation and invasion of glioma cells. These findings enhance our understanding of exosome biology, and may inspire the development of new therapies for glioma.


Abstract
Background: Exosomes are membrane-bound extracellular vesicles of 40-150 nm produced by many cell types, and playing an important role in the maintenance of cellular homeostasis. Exosome secretion allows for the selective removal of harmful substances from cells. Whether this process also takes place in glioma cells is unknown.
Methods: The role of the tumour suppressor miR-375 was explored in human glioma cell lines.
Immunoblotting and qRT-PCR experiments demonstrated a functional link between miR-375 and its target, connective tissue growth factor (CTGF), and led to identify the involved molecular pathways. The exosomes secreted by glioma cells were extracted by ultracentrifugation and examined by transmission electron microscopy. The exosomal expression of miR-375 was analyzed by qRT-PCR. The exosome secretion inhibitor, GW4869, was used to study the biological signi cance of miR-375 release. Moreover, the dynamics of miR-375 release by glioma cells was investigated by using uorescently labelled exosomes. Finally, exosomal miR-375 release was studied in an orthotopic xenograft model in nude mice.
Results: MiR-375 expression was downregulated in gliomas. MiR-375 suppressed glioma proliferation, migration, and invasion by inhibiting the CTGF-epidermal growth factor receptor (EGFR) signalling pathway. MiR-375-containing exosomes were also found in human peripheral blood samples from glioma patients, and their level was correlated with the status of disease progression. Exosomal miR-375 secretion had an impact on the activity of the CTGF-EGFR pathway. Once secreted, exosomal miR-375 was not reuptaken by glioma cells.
Conclusions: Exosomal miR-375 secretion allowed for sustained activation of the CTGF-EGFR oncogenic pathway, promoting the proliferation and invasion of glioma cells. These ndings enhance our understanding of exosome biology, and may inspire the development of new therapies for glioma.

Full Text
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Figure 1 MiR-375 is poorly expressed in glioma tissue and glioma cell lines. a.Calculation of miR-375 expression through dbDEMC2 software. MiR-375 is relatively lower expressed in multiple human maligancies, including glioma. b.Volcano plots of the distributions of miR-375. c. qRT-PCR analysis of miR-375 expression in six glioma cell lines and normal colloidal cells. All experiments repeated independently three times. Data are presented as means±standard deviation. **p < 0.01; ***p < 0.001.

Figure 2
MiR-375 inhibits the proliferation, migration and invasion of glioma cells. a. The expression of mature miR-375 in stable transfectants was con rmed by qRT-PCR. b-c. CCK8 and EdU assay to detect proliferation of miR-375 overexpressing stably transfected U87 and U251 cells. Scale bar, 100 μm. d. Cell migration was determined by a wound-healing assay. The migration distance was measured at 0, 24, and 48 h after the cells were scratched. Scale bar, 400 μm. e. The effect of miR-375 overexpression on the migration (left) and invasion (right) of both U87 and U251 cells examined using a Transwell assay. Scale bar, 100 μm. All experiments repeated independently three times. Data are presented as means±standard deviation. **p < 0.01; ***p < 0.001. ns not signi cant.

Figure 3
CTGF is a direct target of miR-375. a. The correlation of CTGF expression level and glioma grade. CTGF expression levels in glioma in TCGARNA-seq (left), CGGA RNA-seq (right) databases. b. The prognosis e ciency of CTGF in all WHO grade patients in CGGA RNA-seq data. c. The predicted binding sequence of human hsa-miR-375 and its binding site in the 3'-untranslated region (UTR) of CTGF were presented for alignment. d. StarBase v2.0 analysis revealed a negative correlation between miR-375 and CTGF in LGG. e. Expression of candidate target gene CTGF in U87 and U251 cells assessed by qRT-PCR following overexpression of miR-375. f. After miR-375 was overexpressed, the expression of CTGF protein in U87 (left) and U251 (right) cells was analyzed by Western blot. GAPDH was used as an internal control. g.
After miR-375 was overexpressed, the secretion level of CTGF was detected by ELISA. All experiments repeated independently three times. Data are presented as means±standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. ns not signi cant.

Figure 4
MiR-375 regulates glioma proliferation, migration, and invasion through the CTGF-EGFR signalling pathway. After miR-375 was overexpressed in glioma cells, the glioma cells were treated with 200ng/ml CTGF or 20ng/ml EGF for 24 hours. a. Western blot analysis of CTGF, pEGFR, EGFR, pAKT, AKT and MMP9 in U87 and U251 cells. b-c. CCK8 and EdU assay to detect proliferation of U87 and U251 cells. Scale bar, 100 μm. d. Cell migration was determined by a wound-healing assay. The migration distance was measured at 0, 24, and 48 h after the cells were scratched. Scale bar, 400 μm. e. Migration (left) and invasion (right) of U87 and U251 cells detected by Transwell assay. Scale bar, 100 μm. All experiments repeated independently three times. Data are presented as means±standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. ns not signi cant.

Figure 5
Glioma cells-derived exosomes carry a high expression of miR-375. a. Immunoblotting for exosomal markers ALIX, TSG101 and CD9, as well as negative control calnexin. b. Uranyl acetate negative stained TEM images, of exosomes isolated from U87 (left) and U251 cell lines (right). Scale bar, 200 nm. c. The size distribution of the isolated exosomes was analyzed by Zetasizer Nano-Zs. d. Detection of miR-375 relative expression levels in U87 and U251 cells and their derived exosomes by qRT-PCR. e. Detection of miR-375 relative expression levels in miR-375 overexpressing U87 and U251 cells derived exosomes by qRT-PCR. All experiments repeated independently three times. Data are presented as means±standard deviation. **p < 0.01; ***p < 0.001. ns not signi cant.

Figure 6
Exosomal miR-375 secretion affects the proliferation, migration and invasion of glioma cells by affecting the activity of CTGF-EGFR pathway. a. Changes in miR-375 levels in miR-375 overexpressing U87 and U251 treated with or without GW4869 were examined by qRT-PCR. b-c. CCK8 and EdU analysis showed that GW4869 enhanced the inhibitory effect of miR-375 on the proliferation of U87 and U251 cells. Scale bar, 100 μm. d. Wound-healing analysis showed that GW4869 enhanced the inhibitory effect of miR-375 on the migration of U87 and U251 cells. Scale bar, 400 μm. e. Transwell analysis showed that GW4869 enhanced the inhibitory effect of miR-375 on glioma cell migration (left) and invasion (right). Scale bar, 100 μm. f. Western blot analysis of CTGF, pEGFR, EGFR, pAKT, AKT and MMP9 in U87 and U251 cells. GW4869 enhanced the inhibitory effect of miR-375 on CTGF and its downstream genes p-EGFR, p-AKT and MMP9. g. Monensin partially restored the inhibitory effect of miR-375 on CTGF and its downstream genes p-EGFR, p-AKT and MMP9. All experiments repeated independently three times. Data are presented as means±standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. ns not signi cant.

Figure 7
Exosome-released miR-375 is not e ciently re-imported by glioma cells. a. The exosomes of PHK26labeled glioma cells were incubated with U87 or U251 cells for 24 hours. Representative laser confocal images showing that exosomes merged with U87 (up) and U251 (down). Exosomes labeled with PKH26 (red), and glioma cells labeled with GFP (green). Nucleuses stained with DAPI (blue). Scale bar, 10 μm. b. Fluorescence images of miR-375mimic-EXO and glioma cells incubated for 24 hours. Exosomes labeled with PKH26 (red). Nucleuses stained with DAPI (blue). Scale bar, 100 μm. c. The uptake of exosomes by glioma cells was assessed by ow cytometry. Exosomes labeled with PKH26 (red), and glioma cells labeled with GFP (green). d. CCK8 analysis showed that miR-375mimic-EXO did not affect the cell proliferation of U87 and U251 cells. e. Wound healing analysis showed that miR-375mimic-EXO did not affect the cell migration of U87 and U251 cells. Scale bar, 400 μm. f. Transwell analysis showed that miR-375mimic-EXO did not affect glioma cell migration (left) and invasion (right). Scale bar, 100 μm. g. qRT-PCR experiments showed that miR-375mimic-EXO did not affect the content of miR-375 in U87 and U251 cells. All experiments repeated independently three times. Data are presented as means± standard deviation. *p < 0.05; **p < 0.01. ns not signi cant.

Figure 8
Circulating exosomes of patients with glioma contain high levels of miR-375. a. Identi cation of plasma exosomes by TEM. b. Detection of ALIX, TSG101 and CD9 protein expression by western blot. c. The size distribution of the plasma exosomes was analyzed by Zetasizer Nano-Zs. d. miR-375 expression in exosomes in plasma from glioma patients and from non-glioma donors detected by qRT-PCR.

Figure 9
Suppression of exosome secretion enhances miR-375-induced inhibition of glioma proliferation and invasion in vivo. After treating mice with intracranial tumors formed by U87-Luc-miR-375 cells with GW4869 or DMSO, the following experiments were performed. a. Bioluminescence imaging showed the tumor size as days elapsed. b. The luminescent signal intensity of the glioma-bearing mice in two groups were analyzed. c. Evaluation of animal survival was carried out according to Kaplan-Meier analysis. d.

Figure 10
Schematic diagram shows the molecular 1053 mechanism of exosome miR-375-mediated glioma cell proliferation, migration and invasion. MiR-375 is excreted by glioma exosomes, which reduces the content of miR-375 in cells As a result, the inhibitory effect of miR-375 on CTGF is weakened. Therefore, CTGF secretes a large amount of cells and activates the CTGF-EGFR pathway of adjacent and selfglioma cells. As a result, the proliferation, migration and invasion of glioma cells are enhanced.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.