Hypoxic Glioma Cell-Secreted Exosomal miR-301a Activates Wnt/β-catenin Signaling and Promotes Radiation Resistance by Targeting TCEAL7

Abstract

Recent evidence suggests that microRNAs (miRNAs) can be released to the extracellular microenvironment and mediate cell-cell communication through exosomes. The aim of this study was to identify exosomal miR-301a (exo-miR-301a) involved in glioblastoma (GBM) radioresistance and reveal the possible mechanisms. The exo-miR-301a specifically secreted by hypoxic GBM cells could transfer to corresponding normoxia-cultured cells and promote radiation resistance. Hypoxic exo-miR-301a directly targeted TCEAL7 genes, which were identified as a tumor suppressor in GBM malignancy and actively repressed its' expression in normoxic glioma cells. Our studies indicated that TCEAL7 negatively regulated the Wnt/β-catenin pathway by blocking β-catenin translocation from cytoplasm to nucleus. Interestingly, we clarified that the Wnt/β-catenin signaling was activated by miR-301a and TCEAL7 mediated the important procession. The exo-miR-301a was involved in the resistance to radiotherapy, and the effects would be reversed by miR-301a inhibition or TCEAL7 overexpression to regulate the Wnt/β-catenin axis. Here we show that exo-miR-301a, which is characteristically expressed and secreted by hypoxic glioma cells, is a potent regulator of Wnt/β-catenin and then depresses radiation sensitivity through targeting anti-oncogene TCEAL7. The newly identified exo-miR-301a/TCEAL7-signaling axis could present a novel target for cellular resistance to cancer therapeutic radiation in GBM patients.

Keywords: Wnt/β-catenin; exosome; glioblastoma; hypoxia; miR-301a.

Figure 1

miR-301a Is Specifically Expressed and Secreted by Hypoxic GBM Cells and Transferred to Normoxic Glioma Cells via Exosome Secretion High miR-301a (A) and serum exo-miR-301a (B) are strongly associated with HIF-1α protein. (C) ELISA assay for HIF-1α performed in GBM cells. (D) Western blot detected the markers of exosomes. (E) Quantification of exosomes by nanoparticle-tracking analysis. The expressions of HIF-1α (F), miR-301a (G), and exo-miR-301a (H) were detected using western blot and qPCR.

Figure 2

Exosomal miR-301a Derived from Hypoxic Glioma Cells Targets TCEAL7 (A) The inverse correlation of miR-301a with TCEAL7. (B) Schematic representation of the putative binding sites in TCEAL7. (C) mRNA 3′ UTR for miR-301a. pGL3-WT-TCEAL7-3′ UTR-Luc and pGL3-MUT-TCEAL7-3′ UTR-Luc reporters were transfected with As-miR-301a or NC. (D) Western blot analysis was performed to evaluate the expression of TCEAL7 in normoxic and hypoxic glioma cells, which were transfected with As-miRNA-301a. (E) Luciferase reporter co-transfected with exosomes. (F) Hypoxic-derived exosomes decreased the expression of TCEAL7. (G) As-miR-301a prevents augmentation of hypoxic exosomes on TCEAL7 protein downregulation. **p < 0.01.

Figure 3

TCEAL7 Is Frequently Downregulated in GBM Malignancy (A) Immunohistochemical analysis showed the expression of TCEAL7. (B) TCEAL7 expression in glioma and normal brain tissues as detected by qPCR. (C) Kaplan-Meier survival curves analyzed glioma patients who had high or low expression of TCEAL7. (D) Western blot was performed to evaluate the expression levels of TCEAL7. (E) TCEAL7 remarkably inhibited the cell viability of glioma cells. (F) Fewer colonies were observed in TCEAL7 vector than control cells. (G) TCEAL7 vector significantly reduced invasive capacity. (H) TCEAL7 vector induced apoptosis detected by fluorescence-activated cell sorting (FACS) analysis. (I) Inhibition of the growth of U87 xenografts treated with TCEAL7 vector.

Figure 4

TCEAL7 Negatively Regulates the Wnt/β-catenin Pathway by Binding to β-catenin without Suppressing the Stability of β-catenin (A and B) TOP/FOP assay revealed the Wnt/β-catenin transcriptional activity when treated with TCEAL7 vector. (C) Knockdown of TCEAL7 promotes the expression of Wnt target genes. (D) Western blot was performed to evaluate the protein levels of β-catenin. (E) The effect of TCEAL7 overexpression on the stability of β-catenin. Cells were treated with cycloheximide (CHX) (20 mg/mL) for the indicated hours. (F) Interaction between purified TCEAL7 and β-catenin proteins. (G) The interaction between ectopically expressed β-catenin and TCEAL7 was examined using immunoprecipitation assay. (H) The interaction between endogenous β-catenin and TCEAL7 was examined using immunoprecipitation assay. (I) Immunofluorescence detected β-catenin translocation from cytoplasm to nucleus.

Figure 5

Exosomal miR-301a Derived from Hypoxic Glioma Cells Increases Wnt/β-catenin Signaling by Targeting TCEAL7 (A) RNA was extracted from normoxic cells incubated with hypoxic exosomes and analyzed for miR-301a level. (B) RNA extracted from normoxic cells incubated with hypoxic exosomes of different origins for 24 h (or PBS as control) was analyzed for the level of pri-miR-301a or pre-miR-301a. (C) Hypoxic-secreted exosomes were fed to normoxic cells in the presence or absence of 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) (20 μM). After 24 h, RNA extracted from the recipient cells was analyzed for miR-301a level. (D) Glioma cells were treated with miR-301a mimics, TCEAL7 vector, As-miR-301a, or si-TCEAL7, and TOP/FOP assays detected the activity of Wnt/β-catenin signaling. (E) Hypoxic glioma cells were treated with As-miR-301a or TCEAL7 siRNA, and TOP/FOP assays detected the activity of Wnt/β-catenin signaling. (F) Western blot analysis was performed to evaluate the protein levels of β-catenin in the nucleus and cytoplasm.

Figure 6

miR-301a Is Involved in the Radiosensitization of Hypoxic Exosome-Co-cultured Glioma Cells (A) Clonogenic assays of cells treated with 0, 2, 4, 6, and 8 Gy radiation. (B) Cell viability is expressed as a percentage relative to scramble cells. (C) Flow cytometry analysis of apoptosis using annexin V and propidium iodide in cells treated with 4 or 8 Gy. (D) Tumor volume of each group was detected. Solid tumors were weighed after observation in different groups. (E) The expressions of Wnt/β-catenin signaling downstream genes were detected by qPCR. **p < 0.01.