LncRNA GSCAR promotes glioma stem cell maintenance via stabilizing SOX2 expression

Gliomas are the most aggressive type of malignant brain tumors. Recent studies have demonstrated that the existence of glioma stem cells (GSCs) is critical for glioma recurrence, metastasis, and chemo- or radio-therapy resistance. Temozolomide (TMZ) has been used as an initial therapy for gliomas. However, the overall survival time is still limiting due to the lack of effective targets and treatment options. Therefore, identifying novel biomarkers for gliomas, especially for GSCs, is important to improve the clinical outcome in the future. In this study, we identify a human-specific long non-coding RNA (lncRNA, ENSG00000250377), termed GSCAR (glioma stem cell associated lncRNA), which is highly expressed in glioma cancerous tissues and cell lines. We reveal that GSCAR positively correlates with tumor grade. Glioma patients with GSCAR high expression exhibit shortened overall survival time, compared to patients with GSCAR low expression. Furthermore, we show that GSCAR knockdown by shRNAs or antisense oligonucleotide (ASO) reduces tumor cell proliferation, migration and xenograft tumor formation abilities. Mechanistic study shows that GSCAR acts as a ceRNA (competing endogenous RNA) for miR-6760-5p to promote the expression of oncogene SRSF1 (serine and arginine rich splicing factor 1). In addition, GSCAR mediates the protein complex formation between DHX9 (DExH-Box helicase 9) and IGF2BP2 (insulin-like growth factor 2 mRNA-binding protein 2), leading to the stabilization of SOX2 (sex-determining region Y-box 2) mRNA and then the transcriptional activation of GSCAR. Depleting GSCAR reduces SOX2 expression and GSC self-renewal ability, but promotes tumor cell responses to TMZ. These findings uncover that GSCAR/miR-6760-5p/SRSF1 axis and GSCAR/DHX9-IGF2BP2/SOX2 positive feedback loop are critical for glioma progression, which could be used as prognostic biomarkers and therapeutic targets in the future.


Cell migration and invasion assays
To produce a wound, the monolayer cells in 6-well plates were scraped in a straight line with pipette tips. Plates were then washed with PBS to remove detached cells.
Images of the scratches were taken at indicated time points using Nikon inverted microscope (Ti-S). The relative gap width was calculated using GraphPad Prism software. For transwell assay, indicated cells in 100μL serum-free medium were plated in a 24-well plate chamber insert (Corning Life Sciences, Cat. 3422), with the medium containing 10% FBS at the bottom of the insert. For invasion assay, the upper chamber of the insert was pre-coated with Matrigel (Millipore Sigma) before plating cells. After incubation for 48 h, cells were fixed with 4% paraformaldehyde for 1 hour and then stained with 0.1% crystal violet for 30 min. After rinsing with water, migrating or invading cells were imaged and quantified.

Cell flow cytometry assays
For cell apoptosis detection, Annexin V FITC Apoptosis Detection Kit I (556547, BD, China) was used to evaluate the cellular apoptosis following the manufacturer's instructions. For cell cycle analysis, indicated cells were digested and washed with PBS twice and then fixed in 75% alcohol overnight at - 20 °C. The fixed cells were washed and then stained with propidium iodide (PI) staining buffer. Indicated cells were then analyzed by the FACSAria SORP machine (BD, USA).

RNA subcellular fractionation and fluorescence in situ hybridization assay
The nuclear and cytoplasmic fractions were isolated using the NORGEN kit (Cat. 21000, NORGEN, USA). The indicated cells were lysed using cell fraction buffer on ice for 10 min, and after centrifugation at 5000 g for 5 min at 4 °C, the supernatant or the pellet was collected for further cytoplasmic or nuclear fraction purification, respectively. For the RNA fluorescence in situ hybridization (FISH) assay, a Cy3-labeled GSCAR probe was designed and synthesized by RiboBio (China), and the FISH kit (RiboBio,Kit, Cat. C10910) was used to determine the RNA expression pattern following the manufacturer's instructions.

Xenograft tumor formation assay
Indicated tumor cells were subcutaneously injected into 4-5 weeks old male nude mice (Purchased from Vital River Laboratories, Beijing). At the end of the experiments, all mice were sacrificed and the tumors were harvested, weighed, and photographed. Nude mice were monitored every day, the xenograft tumor volumes were measured with a sliding caliper, and tumor volumes were calculated using the formula (L×W 2 )/2. For TMZ and ASO treatment assay in vivo, until the xenograft tumors reached a volume of 50 mm 3 , mice were randomly divided into indicated treatment groups. The nude mice were treated with ASOs (intratumoral injection) with or without TMZ (intraperitoneal injection) every 3 days, PBS intratumoral injection was used as the control group. All mice were sacrificed at the end of the experiment and tumors were harvested, pictured, and weighed. All animals were kept in an SPF environment and the protocols were pre-approved and conducted under the policy of the Animal Care and Use Committee at the Kunming Institute of Zoology, CAS.

Dual-luciferase and RT-PCR assays
For the dual-luciferase assay, indicated DNA fragments were synthesized and cloned into pGL3 basic vector (Table S1), Renilla luciferase plasmid, and indicated plasmids were co-transfected into indicated cells, 24~48 hours later, the luciferase activities were examined by Dual Luciferase Kit (Promega, E1960).
The cDNAs were used for RT-PCR assay using FastStart Universal SYBR Green Master Mix (Roche, 04194194001) and an Applied Biosystems 7500 machine. The detailed primer and oligo sequences used in this study were provided in Table S1.

Immunohistochemical staining (IHC)
Briefly, the tissue sections were deparaffinized in xylene and rehydrated using graded ethanol. Antigen retrieval was performed using sodium citrate buffer (pH 6.0), and the endogenous peroxidase activity was quenched with 3% H2O2, sections pretreated with 1% bovine serum albumin buffer were then incubated with indicated primary antibodies overnight at 4°C. After several washes, the sections were treated with HRP-conjugated secondary antibody for 40 min at room temperature and stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB). Slides were photographed with a microscope (Olympus BX43F, Japan), and representative images were analyzed with the Image-Pro Plus 7.0 software (Media Cybernetics, Inc., Silver Spring, MD, USA). cells using the nuclear and cytoplasmic RNA fractionation assay followed by the RT-PCR examination. β-actin (ACTB) and U1 were used as cytoplasmic and nuclear fraction controls, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001.     Supplementary Tables. S1 to S7