Patients and specimens

The patients were retrospectively selected from 120 consecutive patients ≥ 18 years old with newly diagnosed GBMs operated at Zhongshan Hospital, Fudan University, between January 2008 and May 2018. Selection criteria were (1) ≥ 2 preoperative T1-weighted contrast (gadolinium) enhancing (T1wGd) magnetic resonance imaging (MRI) scans taken ≥ 14 days apart and (2) histopathologically verified diagnosis after the 2021 WHO classification. Exclusion criteria were gliomatosis cerebri and non-contrast-enhancing tumors. The IDH mutation status has been checked with immunohistochemistry for IDH-R132H. The diagnosis of glioma was confirmed by Pathologists. Detailed patient information is presented in Supplementary Table S1.

Cell culture

HEK293T, HEB, U-251MG (U251), U-118MG (U118), U87MG (U87) cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Chinese Academy of Sciences, Shanghai, China). Cells were cultured in DMEM/High Glucose (HyClone, USA) supplemented with 10% FBS and 1% penicillin/streptomycin at 5% CO2 at 37 °C. All cell lines were routinely screened for mycoplasma contamination by MycAwayTM Plus-Color One-Step Mycoplasma Detection Kit (Yeasen, Shanghai, China).

Cell transfection, plasmid construction and lentiviral infection

Cell transfection using Lipo2000™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Construction of the ATP11B overexpression plasmid from the empty vector (pCDNA 3.1) is described in our previous work [24, 25]. For the knockdown of ATP11B, cells were transfected with specific small-interfering RNA (siRNAs) or control siRNA synthesized by RiboBio (RiboBio, GuangZhou, China) using Lipo2000™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The miR-486-3p inhibitor and miR-486-3p mimic were also synthesized by RiboBio. For the knockdown of LINC00606, specific siRNA and control siRNA were synthesized by Tsingke (Tsingke Biotechnology, Shanghai, China). For overexpression, cDNA encoding human ATP11B (NM 014616) in the pCAG-HAC plasmid was kindly provided by Dr. Hye-Won Shin (Kyoto University). Taking the transfection system of six well plates per well cell as an example, the cell seed plate was performed one day before transfection, so that the density of adherent cells reached 50–60% the next day. During the transfection process, the transfection system was configured and the plasmid concentration per well system was 2 μg. The mimic, inhibitor, or siRNA are 50 nM. After 8 h of transfection, the medium was renewed and the cells were incubated for a further 48 h, after which the cells were harvested and used for subsequent experiments. The lentiviral vectors (OE-ATP11B, Vector, sh-NC and sh-LINC00606) were synthesized by Tsingke (Tsingke Biotechnology, Shanghai, China). The siRNA sequences are presented in Supplementary Table S2.

RNA isolation, reverse transcription, and real time PCR

Total RNA was isolated from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was reverse-transcribed in cDNA using Strand cDNA Synthesis (Yeasen, Shanghai, China). Real-time PCR was performed on the cDNA using Yeasen SYBR qPCR Master Mix (Yeasen, Shanghai, China). The Bulge-LoopTm miRNA RT primer and qRT-PCR Starter Kit (Ribobio, C10712-1) was used to detect miR-486-3p expression. GAPDH and U6 were used as an endogenous control for normalization. The relative fold changes in expression were analyzed using the 2−△△CT method. The related primers are given in Supplementary Table S3.

Cell proliferation and colony formation assays

The cell proliferation assay was performed using a Cell Counting Kit-8 (Yeasen, Shanghai, China). Transfected glioma cells (2 × 103) were collected at 48 h post-transfection and seeded onto a 96-well plate. The OD450 nm was measured for 1.5 h after the addition of 10 µL CCK8 solution. For the colony-formation assay, cells were seeded onto a 6-well plate at a density of 1 × 103 cells/well. After 2 weeks, the cells were fixed with 1% crystal violet stain for 20 min at room temperature. The number of visible colonies was counted using the Image J software.

Cell migration and wound-healing assays

Cell migration assays were performed using Transwell chambers (Corning Costar, Tewksbury, MA, USA). A total of 4 × 104 transfected glioma cells in 300 μL serum-free medium were seeded into the upper chamber of each insert, and 500 μL medium supplemented with 10% FBS was added to the lower chambers. After incubation at 37 °C for 24 h, cells were fixed with 1% crystal violet stain for 20 min at room temperature. For wound-healing assays, 4 × 105 cells/well were seeded onto 24-well plates and cell growth was recorded at 0 h and 24 h. The cell migration rate was analyzed using the Image J software.

Flow cytometry

The Apoptosis Detection Kit was used (Yeasen, Shanghai, China) to detect apoptosis levels in transfected cells. Cells were resuspended in 50 μL 1 × binding buffer, and 2.5 μL Annexin V-FITC or 5 μL anti-PI-PE were added to the reaction for 15 min at room temperature. Subsequently, 1 × binding buffer (300 μL) was added to each sample. Data were collected by flow cytometry (Beckman Coulter Cytoflex).

CHIRP

The probe for CHIRP was synthesized in RiboBio, and the CHIRP experiment was carried out according to the instructions of CHIRP kit (BersinBio, Bes5104, Guangzhou, China). CHIRP experiments were conducted in accordance with BersinBio’s CHIRP Kit (China) operating guidelines, which are detailed below. (1) Cross-link Cells, digest cells with trypsin, the cell count is 1.2 × 108, add 30 mL of 1 × PBS (containing 810 µL of 37% formaldehyde, formaldehyde final concentration 1%) to each tube of samples, cross-link at room temperature for 10 min with a vertical mixer; then mix with 1.375 M Glycine for 5 min; 4 °C, 1500 rpm, collect cells by centrifugation for 5 min, discard the upper layer of PBS solution, and repeat twice; (2) Cell Lysis, the sample is placed on ice, and the lysate and reselection solution in the kit are configured to perform the lysis operation on the sample. (3) Sonication, under ice bath conditions, 3 s ON, 5 s OFF, 230W power ultrasound for 20 min until the sample is clarified; (4) Preclearing, after ultrasound, the sample was added to Agarose beads, and the binding was incubated at 4 °C for 60 min on a vertical mixer; Input (protein, RNA) was reserved and 3 mL of sample was transferred, labeled as NC, odd, even, respectively. (5) Probe preparation, in which the LINC00606 probe is divided into odd and even groups. According to the operating instructions, the LINC00606 exceeds 1200 nt. The probe group is divided into two groups. The odd number is the odd group (indicating the number of the first odd probe), and the even number is the even group (indicating the number of the first even probe). Take 8 µL of odd, even and NC probe group solutions (80 pmol probe/strip/1 mL sample); After 3 min of denaturation at 85 °C, quickly transfer the ice bath. (6) Probe preparation, prepare Streptavidin Beads according to the ratio of 50 μL beads/200 pmol probes. (7) CHIRP, configure the sample-lysis hybridization system, incubate and denature at 65 °C for 10 min on a mixer, then add the probe to the denatured sample, hybridize at 37° C for 30 min, incubate and denaturate at 50 °C for 5 min, and hybridize at 37 °C for 90 min; add the CHIRP sample-probe group samples to the magnetic bead mixture respectively, and incubate and bind on a vertical mixer at room temperature for 30 min; then perform the cleaning step, the magnetic beads are collected by the magnetic frame and supernatant is removed; the samples are collected and then followed by CHIRP-RNA Isolation; CHIRP-Protein sample collection and other experiments. Details of the sequences are listed in Supplementary Table S4.

Fluorescence in-situ hybridization (FISH)

A specific fluorescently labeled LINC00606 FISH probe was designed and synthesized by Servicebio (Wuhan, China). Fluorescence-labelled single-strand probes were hybridized. The FISH experiment was performed according to the SweAMI-FISH manufacturer’s instructions (Servicebio). In detail, we take the cell sample as an example and fix it using 4% paraformaldehyde (DEPC configuration) for 20 min, wash it with PBS three times, then digest the sample dropwise with proteinase K (20 μg/mL) for 5 min, then wash it. Treat the sample using the hybridization solution (Servicebio China) and place it in a 37 °C incubator for 1 h. Then treat the sample using the hybridization solution (containing LINC00606-FISH-probe at a concentration of 500 nM) and place it in a 40 °C hybridization oven overnight. The washing process consists of washing the SSC solution (2 × , 1 × , 0.5 ×) with different layers of concentration for 5–10 min respectively. Dilute the signal probe (1 ×) (Servicebio China) using the hybridization solution and then place it in a wet box at 40 °C for hybridization for 50 min, followed by the cleaning steps. Repeated hybridization and washing of the signal probe were performed to enhance the signal of the probe. Immunofluorescence staining was performed afterwards, and the samples were sealed and broken. Blocking was performed using 5% goat serum. The broken membrane was permeated with 1 × PBS containing 1% goat serum and 0.25% Triton ™ X-100, and then stained with ATP11B antibody overnight at 4 °C. The next day, the climbing tablets were washed 3 times with 1 × PBS for 3 min each time, incubated with fluorescent secondary antibody for 1 h at room temperature, and then washed again. DAPI (Invitrogen, NBP2311561) was used to counterstain the nuclei after incubation for 5 min in the dark. Finally, samples were washed four times with PBST for 5 min each to remove excess DAPI, and then sealed with a sealing solution containing a fluorescent quencher. All fluorescence images were captured using a laser confocal microscope (Zeiss, Germany). Details of the sequences are listed in Supplementary Table S4.

Immunoblotting

Immunoblotting analysis was performed as described previously [26]. In detail, wash the cells with pre-cooled PBS and lyse them on ice with Western and IP lysate (Beyotime, China) for 15 min. Collect the protein lysate into an EP tube, centrifuge at 12,000 rpm for 30 min at 4 °C, collect the supernatant, and determine the soluble protein concentration using a BCA protein detection kit (Sparkjade, China). Add protein lysate to 5 × loading buffer (Beyotime, China) and boil for 10 min for SDS-PAGE (6–15%) electrophoresis, then transfer to NC or PVDF membranes. The membranes were sealed with 5% skim milk at room temperature for 2 h, incubated overnight with primary antibody at 4 °C (Abclonal, Wuhan, China), and then incubated with secondary antibody at room temperature for 1 h. Visualization of protein bands using the Tanon gel imaging system. Relative protein levels are normalized to GAPDH or Vinculin. Antibodies used in this study are shown in Supplementary Table S5.

Dual luciferase reporter assay

Verify the relationship between LINC00606 and miR-486-3p or miR-486-3p and TCF12 using the Dual Luciferase Reporter Assay Kit (Vazyme, China). Taking miR-486-3p and TCF12 as examples, we predicted the binding sequence of miR-486-3p and TCF12 through RNA hybridization software and analyzed the free energy of its targeted binding checkpoint. We designed the mutant (MUT) binding checkpoint using the wild-type (WT) TCF12 complementary sequence to construct pGL3 Basic reporter plasmid (Tsingke Biotechnology, Shanghai, China). Transfect 293T cells with TCF12-MUT (mutant 3'UTR) or TCF12-WT (luciferase reporter plasmid, sequence correct) and miR-486-3p mimic or NC (Ribo, China) for 48 h. After the cells were lysed using the lysate in the kit, the supernatant was centrifuged at 12,000 rpm for two min for subsequent experimental detection. Luciferase substrate, cell lysate, and renilla substrate working fluid were added sequentially to the enzyme label plate, and each group was detected separately using an enzyme label instrument. Relative luciferase activity was the ratio of firefly luciferase to renal luciferase. Three independent experiments were carried out. Details of the sequences are listed in Supplementary Table S6.

RIP

We take the cell sample as an example, wash it twice with pre-cooled PBS, then use cell scraping to collect the cells, and centrifuge to collect the cells at 4 °C to obtain the cell pellet. Add lysate (Beyotime, China) to lyse the cells, and centrifuge to take supernatant and wait for follow-up experiment. Wash the protein A/G magnetic beads (ABclonal, RM02915, Wuhan, China) with NT2 buffer (containing 50 mM Tris–HCl, 150 mM NaCl, MgCl2 1 mM, 0.05% NonidetP-40, ddH2O), and spin the corresponding antibody ATP11B (5 μg) at room temperature for 2 h. Then collect the magnetic beads in a magnetic rack, wash and set aside. Configure the immunoprecipitation buffer (800 μL NT2 buffer, 35 μL 0.5 M EDTA, 5 μL RNase inhibitor), add the collected magnetic beads and cell lysate to the immunoprecipitation buffer and transfer to a rotator overnight at 4 °C. The reserved input group is the cell lysate. For the magnetic bead sample, perform the cleaning step. The sample is RNA purified, simply put the magnetic bead sample into the protease K solution (containing 117 μL NT2 buffer, 15 μL 10% SDS, 8 μL 10 mg/mL protease K), shake at 55 °C for 30 min, and collect the supernatant for subsequent RNA extraction experiments.

RIP-qPCR

Protein A/G magnetic beads for 1 h (ABclonal, RM02915, Wuhan, China) were washed with NT2 buffer and incubated with anti-IgG or anti-Argonaute2 for 1 h. Finally, the magnetic bead-antibody complex was rinsed with RIP wash buffer and incubated with protein lysate overnight at 4 °C to evaluate the binding of Argonaute2 (AGO2) to LINC00606 and miR-486-3p. The magnetic bead-protein-RNA complex was reconstituted in purification solution (10 mg/mL protease K). Total RNA was obtained using TRIzol for subsequent qPCR detection. Details of the antibodies are listed in Supplementary Table S3.

RNA pulldown

Reference RNA Labeling Mix (Roche Life Science, Germany) was used for biotin labeling of RNA, according to the manufacturer’s instructions. LINC00606 was labeled with biotin-16-UTP by in vitro transcription with T7 RNA polymerases, and RNA was purified using an RNAclean Kit DP412 (TIANGEN, Beijing, China). Biotinylated RNA was incubated with U251 lysates for 1 h at 4 °C, following which Streptavidin beads (Abclonal, RK20270, Wuhan, China) were added and further incubated overnight at 4 °C. The next day, the Streptavidin beads were washed with PBST (0.5% Tween-20), and the enriched proteins were collected for immunoblotting.

CHIP-qPCR

The CHIP assay has been described previously [27]. U251 cells were fixed with 1% formaldehyde and collected in lysis buffer. TCF12 and IgG were used for CHIP. Immunoprecipitated DNA was amplified by PCR using specific primers (Supplementary Table S3).

Tumor xenograft model

All animal experiments were carried out following NIH Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care Committee of Shanghai University. Cells were resuspended in 2 μL matrigel and injected into the nude mouse (Beijing Vital River Laboratory Animal Technology Co., Ltd.) brain using stereotaxic apparatus. Injection location: parietal-occipital area of mouse brain; injection coordinates 0.5 mm, -2 mm, -3 mm. Four-weeks post-injection, nude mice were sent to Huashan Hospital Affiliated to Fudan University, where the tumor marker 18F-fluoroethyl-tyrosine (18F-FET) was injected into nude mice through the tail vein and the Siemens Inveon PET/CT system was used to evaluate tumor formation in the brain. These nude mice were randomly divided into 4 groups (6 in each) for different treatments: sh-NC, sh-LINC00606, OE ATP11B, and Vector. A total of 4 × 106 transfected U251 cells were injected subcutaneously into mice and growth of the resulting tumor was recorded. Mice were sacrificed after 25 days, and the volume and weight of the tumors were measured. Tumor volume was calculated as volume = length × (width)2/2.

Statistical analysis

Image J, GraphPad Prism 8, Zesis, Image studio Ver 5.2, GEPIA2 (gepia2.cancer-pku.cn) were applied for analysis. The means between different groups were analyzed using a Student’s t-test and one-way analysis of variance. Data are expressed as the mean ± standard deviation (SD) unless otherwise noted, with a minimum of three replicates. Statistical significance was set at P < 0.05.