Hypoxia-Induced Glioma-Derived Exosomal miRNA-199a-3p Promotes Ischemic Injury of Peritumoral Neurons by Inhibiting the mTOR Pathway

The underlying molecular mechanisms that the hypoxic microenvironment could aggravate neuronal injury are still not clear. In this study, we hypothesized that the exosomes, exosomal miRNAs, and the mTOR signaling pathway might be involved in hypoxic peritumoral neuronal injury in glioma. Multimodal radiological images, HE, and HIF-1α staining of high-grade glioma (HGG) samples revealed that the peritumoral hypoxic area overlapped with the cytotoxic edema region and directly contacted with normal neurons. In either direct or indirect coculture system, hypoxia could promote normal mouse hippocampal neuronal cell (HT22) injury, and the growth of HT22 cells was suppressed by C6 glioma cells under hypoxic condition. For administrating hypoxia-induced glioma-derived exosomes (HIGDE) that could aggravate oxygen-glucose deprivation (OGD)/reperfusion neuronal injury, we identified that exosomes may be the communication medium between glioma cells and peritumoral neurons, and we furtherly found that exosomal miR-199a-3p mediated the OGD/reperfusion neuronal injury process by suppressing the mTOR signaling pathway. Moreover, the upregulation of miRNA-199a-3p in exosomes from glioma cells was induced by hypoxia-related HIF-1α activation. To sum up, hypoxia-induced glioma-derived exosomal miRNA-199a-3p can be upregulated by the activation of HIF-1α and is able to increase the ischemic injury of peritumoral neurons by inhibiting the mTOR pathway.


Primary neuronal cultures
Rats were anesthetized with isoflurane and the E18 embryos were removed. The cortical region of the fetal brains was dissected in warm media and pooled together. The cortices were triturated and incubated in papain for 20 min at 37 °C, then centrifuged at 1500 rpm for 5 min at room temperature (RT). Cells were resuspended in MEM (minimal essential medium) (Gibco, Grand Island, NY, USA) containing 10% fetal horse serum (Hyclone, Logan, UT, USA), 2 mmol/l glutamine (Gibco), 25 mM glucose, and 1% penicillin/streptomycin (Gibco). Cells were plated onto poly-D-lysine-coated tissue culture plates at 7.5×10 5 cells/ml. Media were completely changed after 24 h. One-half medium changes were performed at day 4. Cultures were incubated at 37 °C in a 5% CO2 incubator and experiments were performed after days 9-11.

Oxygen-Glucose Deprivation (OGD) and Reperfusion
Aspirate the former medium in the 6-well plates 9hformer medium to simulate ischemia; the control should be disposed with BSS5.5 and incubated in 37°C, 5% CO2. Then place the OGD-R groups in the Hypoxia Incubator Chamber, and 10-20 mL 2-time sterile H2O to maintain the proper humidity within the chamber. Lock the clamp and open the inlet of the chamber and link the bacteria filter with the outlet tube of N2 Bottle. Next, open the switch and modulate the flow volume to 20L/min and sustain the nitrogen flow for 5min and then block the outlet and inlet of the chamber to maintain the low O2 pressure within the chamber. Place the chamber into the 37°C incubator for 6 hrs. and control groups are incubated directly in the 37°C, 5%CO2 incubator.
Fetch the OGD groups and aspirate the former BSS0. Well rinse twice with BSS5.5, and add the same volume of it as former BSS0 into wells. Incubate the OGD groups and control in the 37°C, 5%CO2 incubator for 6 hrs.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
For the purposes of the experiments at the end of the incubation time, cells were incubated for 4 h with 0.8 mg/ml of MTT, dissolved in serum free medium (MEM or DMEM for HepG2 and HTC cells respectively). Washing with PBS (1 ml) was followed by the addition of DMSO (1 ml), gentle shaking for 10 min so that complete dissolution was achieved. Aliquots (200 μl) of the resulting solutions were transferred in 96-well plates and absorbance was recorded at 560 nm using the microplate spectrophotometer system (Spectra max190-Molecular Devices). Results were analyzed with the Soft max pro software (version 2.2.1) and are presented as percentage of the control values. The materials were from Sigma Aldrich unless otherwise stated.

Transmission electron microscopy (TEM)
After the exosome samples were prepared as previously described, the purified exosomes were re-centrifuged using an Exosome Isolation Kit to collect exosome pellets. Briefly, each exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4°C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatin, fixed in glutaraldehyde at 4°C and cut into small blocks (less than 1 mm3). The samples were dehydrated in increasing concentrations of alcohol, placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin, which was allowed to polymerize at 35°C for 12 h, 45°C for 12 h, and 60°C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (NanoSight Ltd., Amesbury, UK) at a voltage of 110 kV.

Lentiviral vectors generation, titration and gene delivery
We used a 3 plasmids system for lentivirus packaging as detailed in our previous study, which include the lentiviral backbone containing miR-199a-3p shRNA and scramble shRNA genes, the packaging plasmid (p-delta) that provides all vector proteins driven by the trip CMV promoter, except the envelope protein, and the envelope-encoding plasmid (p-VSVG) that encodes the heterologous vesicular stomatitis virus envelope protein (VSVG). In brief, a mixture of 45 μg of transfer vectors, 30 μg of packaging plasmids and 15 μg of envelopeencoding plasmids were transiently transfected into 3 T175 flasks containing 1.5 × 107 HEK-293T cells using the calcium phosphate precipitation (CPP) method. Supernatants were collected 72 h post-transfection and viral particles were concentrated by ultracentrifugation. Viruses were resuspended in phosphate-buffered saline (PBS) and kept at −80 °C until use.
Virus titers ranged from 2 × 10 8 to 5 × 10 8 TU/ml and were diluted in PBS before gene transfer was conducted. The lentiviral vectors of GFP , S6K, HIF-1a, miR-199a-3p shRNA, scramble shRNA and diluted with PBS, were directly added in the medium of HT22, C6 cell cultures or primary neurons (10, 5 and 2 μl vectors were added into each well of 6, 24 and 96 well plates, respectively) with the multiplicity of infection at 1:5 (cells to virus units) immediately after medium exchange, and then incubated for 0-6 days. The same amount of PBS was also added to the medium as the experimental control (100 %).

Western blotting
Thirty micrograms of protein in each lane was subjected to SDS-PAGE using 4-15% Ready Gel (catalog #L050505A2; Bio-Rad, Hercules, CA) under 200 V for 45 min. Protein bands were transferred from the gel to polyvinylidinene fluoride (Millipore, Bedford, MA, USA) membranes under 100 V for 2hr. After blocked with Tris buffered saline containing 5%(w/v) bovine serum albumin and 0.1% Tween-20, the memberane was incubated with primary antibodies (supplementary table 1) incubated overnight at 4 °C followed by Alexa Fluor 488 donkey anti-rabbit or anti-mouse IgG secondary antibody (1:5 000, Invitrogen, Eugene, OR, USA) for 1hr in the dark room. Then membranes were scanned using Typhoon trio (GE Healthcare). The optical densities of all protein bands were analyzed using IMAGEQUANT 5.2 software (GE Healthcare). All samples were run on the same gel. The protein bands were rearranged solely to ease comparison in figures.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized from 0.5 μg total RNA by reverse transcription using an ImProm-II reverse transcription kit (Promega, Madison, WI, USA) with random hexamer primers. Quantitative real-time PCR was performed using the SYBR Green QPCR system (Qiagen, Valencia, CA, USA) with specific primers. The PCR reactions were performed using an Applied Biosystems Prism 7000 sequence detection system. The level of target genes expression was normalized against the GAPDH gene. Data were analyzed with SDS 2.2.2 software using the 2 -ΔΔCt method with a relative quantification RQmin/RQmax confidence set at 95%.  5′-ACTTCGGGTACTTGGTAAAGG-3′ 5′-GATGTTCTCCGGCTTCA-3′