Clinical sample preparation and high-throughput sequencing analysis
A total of 144 glioblastoma samples and 8 normal brain samples (normal cortical brain tissue obtained during glioma surgery) were collected from the Department of Neurosurgery of Shandong Provincial Hospital affiliated with Shandong University. The research was approved by the Research and Ethics Committee of Shandong Provincial Hospital.
To detect the candidate lncRNAs in human GBM tissues, total RNA was first extracted with TRIzol Reagent (Invitrogen, CA, USA) from the tissues of five GBM patients (five GBM samples, four paracancerous samples and five normal brain samples in total). Labeling and hybridization to the Affymetrix GeneChip Human Exon Arrays-Gminix- lncRNA-WT (Gminix, Shanghai, China) based on extracted RNA were then conducted. The lncRNAs were carefully constructed using publicly available transcriptome databases (RefSeq, UCSC Known Genes, Ensemble, NONCODE, etc.). After quantile normalization of the raw signal, 249 lncRNAs with significant differences among GBM, paracancerous and normal brain tissues (FDR<0.01) were chosen for further data analysis. For RNA-seq analysis, mRNA sequencing was conducted using the Illumina HiSeq Platform (PE150). Briefly, total RNA was isolated and subjected to cDNA library construction, in which the clean (high-quality) reads were aligned to the Human Genome Reference (GRCh38). Gene expression normalization was performed by Fragments per Kilobase per Million Mapped Fragments (FPKM). Using a high-throughput sequencing technique (Affymetrix GeneChip® Human Transcriptome Array 2.0), we defined clusters of differentially expressed lncRNAs in glioma specimens and paracancerous tissues. The suffix E-Signal represents the paracancerous tissues, and the suffix T-Signal represents the tumor tissues. Clusters in green indicate the downregulated lncRNAs, and clusters in red indicate the opposite.
Cell culture and reagents
The high-grade human glioma cell lines U251 and U87 were obtained from American Type Culture Collection (Manassas, VA, USA) and used for in vitro experiments. The normal human astrocyte (NHA or HA) cell line was obtained from ScienCell (Carlsbad, CA, USA). Tumor cells were maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin. The culturing environment was 37 °C with 5% CO2. GSCU251 (Glioma stem cell derived from U251) were obtained from the U251 cells cultured in DMEM/F12 medium supplemented with 2% B27, 25ng/ml human recombinant basic fibroblast growth factor, 25ng/ml epidermal growth factor and 1% penicillin/streptomycin. Half of the sphere-forming medium was replaced every other day.
Reagents, antibodies and MDHDH sequence information
Detailed information on the specific reagents and antibodies is listed in Supplementary Table 1. LncRNA MDHDH reference sequence information is listed in Supplementary Table 2, and NCBI-ORFinder potential peptide alignment results are listed in Supplementary Table 3.
RNA chromogenic in situ hybridization (CISH) and RNA fluorescence in situ hybridization (FISH) assay
To detect the expression status of MDHDH in different grades of glioma, a tissue microarray obtained from Outdo Biotech Co., Ltd. (S/N: HBraG090PG01-M-081, WHO grade 1-4 and normal brain tissue) was employed. The MDHDH-CISH probes were designed and synthesized by Boster Biological Technology Co., Ltd. The CISH assay kit was obtained from the same company and applied according to the manufacturer’s protocol.
In addition, to detect the subcellular localization of lncRNA MDHDH, a Fluorescence in Situ Hybridization Kit (RiboBio, Guangzhou, China, FISH probe sequences listed in Supplementary Table 4) was utilized. The probes were designed and synthesized by RiboBio, Guangzhou. U87 and U251 cells were used in the experiments.
Transfection of the MDHDH smart silencer
RiboBio lncRNA Smart Silencer was provided by Guangzhou RiboBio Co., Ltd. (the smart silencer sequences are listed in Supplementary Table 4). Transfection was conducted according to the manufacturer’s protocol. Briefly, cells were grown to 30-50% confluence and transfected with oligonucleotides mixed with Lipofectamine 2000 at a concentration of 100 nM. The smarter silencer was represented as the pool containing three siRNAs and three antisense oligonucleotides that target different sites of MDHDH.
Vector construction and small interfering RNA (siRNA)
To generate a GBM cell model that is close to the basal MDHDH expression level of normal brain tissue, we synthesized the MDHDH sequence (NR_028345) via Biosune (Shanghai, China) and cloned it into the eukaryotic expression vector (pcDNA3.1) (Invitrogen, CA, USA). This vector possesses the restriction endonuclease site XhoI, which can be used to generate RNA probes for the subsequent RNA pull-down experiments.
We obtained the three siRNAs of PSMA1 from GeneralBiol (Anhui, China). To confirm whether the molecular scaffold function and phenotypic regulation function of MDHDH are dependent on PSMA1, siRNA (5’-GGGCAGGAUUCAUCAAAUUTTAAUUUGAUGAAUCCUGCCCTT-3’) duplexes with proven knockout efficiency and targeting human PSMA1 (PSMA1 siRNA) were used to transfect GBM cells. The siRNA transfection was performed using the transfection reagent Lipofectamine RNAiMAX (Invitrogen, CA, USA) according to the manufacturer’s protocol. Briefly, the siRNA was added to each well at a final concentration of 100 nM. Six hours later, the medium was replaced with DMEM containing 10% FBS, and the cells were incubated for 72 h. The PSMA1 expression levels were determined by qRT–PCR and Western blotting.
Quantitative reverse-transcription polymerase chain reaction (qRT–PCR)
Briefly, total RNA was extracted from the cell lysates, cytoplasm extracts or nuclear extracts by TransZol Up (Transgen, Beijing, China). RNA was quantitatively analyzed using a Nanodrop (Nanodrop Technologies, Rockland, DE, USA). Total RNA (1 μg) was reverse transcribed into cDNA using a cDNA synthesis kit (Transgen, Beijing, China) according to the manufacturer’s instructions. RT–PCR was performed in a Bio-Rad CFX connect real-time system detector with Transgen SYBR Green Supermix. The reactions were analyzed using Bio-Rad CFX Maestro software (Version 4.1). The threshold cycles (CT) were calculated, and the relative gene expression was analyzed after normalizing to beta-actin. The experimental primers were designed and produced by Takara (Japan) and are listed in Supplementary Table 5.
Cell viability assay
The Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc. Beijing, China) was used to determine cell viability. After overexpression or RNA silencing, cells were seeded at 1×103 cells/well in 96-well plates. At different time points, the culture medium was replaced with 100 μL of fresh medium containing 10 μL of CCK-8 solution. The cells were further incubated for 2 h at 37 °C , and the optical density (OD) at 450 nm was measured. Each experiment was repeated three times.
Wound-healing and transwell assays
A wound healing assay was performed in 6-well cell culture plates (Corning, USA). The scratching step was performed vertically on the center of each well using 200 μL pipettes, and the scratched cells were cultured for 12 h and 24 h in high-glucose DMEM without fetal bovine serum (FBS). Images were obtained using a microscope at 200×, and the gap distance was measured by the plotting scale of the software. The proportion of changes was calculated and statistically analyzed. Transwell assays were applied to evaluate the invasion and migration capacities. Glioma cells were seeded in transwell chambers (24-well format; Corning, USA). For the invasion test, we used Matrigel-coated chambers (BD Biosciences, NJ, USA). Cells were cultured with 100 μL serum-free DMEM in the upper chamber for 6 h, and the lower chamber containing 500 μL medium was supplemented with 20% FBS for transwell tendency. Then, the cells in the upper transwell chambers’ supernatant and attachments were removed. Cells on the bottom surface of the chambers were fixed in methanol for 5 min, stained with crystal violet (Solarbio, Beijing, China) and counted in three random fields under a microscope. Each experiment was repeated three times.
In brief, the cells were harvested and lysed with protein extraction agent (RIPA, Solarbio, Beijing, China). Considering the specific application, a Nucleus and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) was utilized. A total of 25-50 μg of protein per sample per lane was mixed with loading buffer (Epizyme, Shanghai, China) and loaded for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%-12.5% SDS–PAGE gel preparation kit, Epizyme, Shanghai, China). Primary antibodies were incubated overnight at 4 °C . Secondary antibodies (goat anti-rabbit and mouse IgG-HRP, Abmart, Shanghai, China) were incubated for 1-2 h at RT. The proteins were visualized using chemiluminescence (ECL) (Affinity Biosciences, OH, USA) and a detection system (Tanon 4800, Tanon, Shanghai, China). All primary and secondary antibody information is listed in Additional file 1.
RNA segmentation, biotin-labeled RNA pulldown and mass spectrometry (MS) assay
Human MDHDH cDNAs (sense and antisense; Biosune Biotech, Shanghai, China) and truncated constructs were transcribed in vitro using the MEGAscript T7 Kit (Thermo Fisher Scientific, MA, USA). The full-length transcript of MDHDH is 746 nt in length; Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, Δ1-del, Δ2-del and Δ5-del correspond to nt 1–293, 294–564, 565–749, 1-49/495-577, 50-494, 578-749, 1-47/181-293, 390-564 and 181-293/390-494 of MDHDH, respectively. For plasmid extraction, an OMEGA Endo-Free Plasmid Mini Kit II (OMEGA BioTek, Guangzhou, China) was applied. For restriction enzyme digestion, FastDigest XhoI (Thermo Fisher Scientific, MA, USA) was utilized. Agarose gel electrophoresis images were obtained by Quantity One software. For DNA purification, we utilized a TIANquick Mini Purification Kit (Tiangen Biotech, Beijing, China). The 3’ ends of the resultant transcripts were labeled with biotin using the Pierce™ RNA 3’ End Desthiobiotinylation Kit (Thermo Fisher Scientific, MA, USA) to generate RNA probes for RNA pulldown, which was performed using the Pierce™ Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific, MA, USA) following the manufacturers’ guidelines. Eluted proteins were detected by mass spectrometry analysis (MS). MS was utilized to identify the proteins that interacted with full-length and/or truncated RNA probes. The filter-aided sample preparation (FASP) method was used for sample preparation . Proteins were digested with trypsin (0.4 μg/μL, Promega) overnight. Peptides were desalted and concentrated using C18-based solid phase extraction prior to analysis by high resolution/high mass accuracy reversed-phase (C18) nano-LC–MS/MS. All raw files were processed using Proteindiscover software (version 1.4, Thermo Scientific) for database searching. MS/MS spectra were searched in the UniProtKB/Swiss-Prot human database. MS analysis was performed by the Advanced Medical Research Institute of Shandong University, and the protein list obtained from the RNA pulldown assay is shown in Supplementary Table 12. After PPI (protein–protein interaction) and gene or pathway enrichment (MCODE) analyses , proteins that may interact with RNA were verified by Western blot.
RIP, Co-IP and ChIP Assays
U87 and U251 cells were seeded, transfected and lysed with lysis buffer (RNase inhibitor included) using an RNA immunoprecipitation Kit (Geneseed, Guangzhou, China), producing a mixture containing bait-target complexes and other irrelevant proteins. Then, a capture antibody and protein A/G magnetic beads were added to the mixture to specifically bind bait protein after antibody-bead crosslinking. According to the manufacturer's instructions, two kinds of columns were utilized to harvest the RNA and proteins bound to magnetic beads. For RIP-seq analysis, RNA sequencing was conducted by the application of Illumina HiSeq Platform (PE150). Briefly, total MDH2-interacting RNAs were isolated and subjected to a library construction, in which the clean (high-quality) reads were aligned. All identified lncRNAs referenced in Supplementary Table 16. Meanwhile, MDHDH-specific primers were used to conduct qRT–PCR analysis on the retrieved RNA. We detected both total RNA (input controls) as well as normal mouse IgG controls to verify that the previously detected signals specifically originated from the MDH2- or PSMA1-binding RNAs. The eluted protein sample (eluted from anti-PSMA1, anti-MDH2 and mouse IgG antibodies) was added to the loading buffer and heated at 95 °C for 15 min for SDS–PAGE analysis. Anti-MDH2 or anti-PSMA1 primary antibodies were used to incubate the transferred PVDF membrane and detect whether the binding degree of PSMA1 and MDH2 changes with the expression status of MDHDH.
U87 and U251 cells were grown to 80% confluence. ChIP assays were performed with a SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, #9003 MA, USA) according to the manufacturer’s directions. Native chromatin immunoprecipitation was performed with an anti-H3K27me3 antibody (Cell Signaling Technology, #9733, 1:50) or anti-H3 (Cell Signaling Technology, #4620, 1:50). Rabbit IgG was employed (ABclonal, AC005, 5 μg) as a negative control. qPCR analysis was performed to detect the DNA fragments immunoprecipitated with H3K27me3. The primer pairs are listed in Supplementary Table 5.
Cell bioenergy tests
Transfected U87 and U251 cells were seeded in XFe 96-well microplates (5×103 cells per well) (Agilent Technologies, Sana Clara, USA) for 24 h. Cells were rinsed and incubated in base medium (Agilent Technologies) at 37 °C for 1 h. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured in real time with a Glycolysis Stress Test Kit and Mito Stress Test Kit, respectively, using a Seahorse XFe96 Analyzer (Agilent Technologies) following the manufacturer’s instructions. Data were normalized by the cell number.
Transfection with adenovirus expressing the mCherry-GFP-LC3B fusion protein
To detect changes in autophagic flux, an adenovirus expressing the mCherry-GFP-LC3B fusion protein (Ad-mCherry-GFP-LC3B) was obtained from Beyotime, China. Cells grown to approximately 70% confluence were transfected with Ad-mCherry-GFP-LC3B according to the manufacturer’s instructions. Ad-mCherry-GFP-LC3B–transduced glioma cells were transfected with different plasmid constructs, visualized by fluorescence microscopy and quantified with FIJI software. DMEM high-glucose medium (with FBS) was replaced with Earle's Balanced Salt Solution (with Ca2+& Mg2+) (EBSS) to induce GBM cell autophagy as a positive control. When autophagosomes accumulated, both red and green LC-3 puncta were observed. The merging of the two channel images generated a yellow signal, which indicated that the autophagy process was not completed (autophagosomes had not yet fused with lysosomes to form autolysosomes). The change in fluorescence signal represented the formation of autophagosomes and the process of autophagosome-lysosome association (yellow- no autophagy; green puncta- autophagosome formation; red puncta- autophagy lysosome formation).
Mitochondrial membrane potential (ΔΨm) assay
A JC-1 probe was employed to measure mitochondrial depolarization in GBM cell lines. Briefly, cells cultured in six-well plates after the indicated treatments were incubated with an equal volume of JC-1 staining solution (5 pg/ml) at 37 °C for 20 min and rinsed twice with PBS. The mitochondrial membrane potential was monitored by determining the relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates using an Olympus fluorescence microscope. Mitochondrial depolarization is indicated by an increase in the green/red fluorescence intensity ratio.
Transfected U87 and U251 cells were collected to determine the NAD+ levels using an NAD+/NADH assay kit with WST-8 (Beyotime, Shanghai, China) and a Mitochondria/Cytosol Fractionation Kit (BioVision, K256-100 CA, USA) according to the manufacturer’s instructions. In brief, cells (1×106/sample) were lysed and isolated with a Mitochondria/Cytosol Fractionation Kit. To measure the total NAD+/NADH ([NADtotal]), 20 μL of cyto-lysates was added to a 96-well plate. To measure NADH ([NADH]), the lysed cells were incubated at 60 °C for 30 min, and 20 μL was added to a 96-well plate. Subsequently, 90 μL of alcohol dehydrogenase was added and incubated at 37 °C for 10 min. Finally, 10 μL of chromogenic solution was added to the plate, and the mixture was incubated at 37 °C for 10 min. A standard curve was generated and measured at the same time as the samples. The absorbance values were measured at 450 nm and analyzed on a plate reader. The amount of NAD+ was derived by subtracting NADH from the total NAD+/NADH ([NAD+] = [NADtotal] - [NADH]; [NAD+]/[NADH] = ([NADtotal] - [NADH])/[NADH]).
Extracellular pyruvate and lactic acid assays
For the pyruvate assay, we utilized a pyruvate testing kit (Jiancheng Bioengineering Institute, Jiangsu, China). We mixed 100 μL of the specimen with 1 mL of the assay reagent in the kit, added 100 μL of the standard pyruvate (0.2 μmol/ml) and 100 μL of double-distilled water to 1 mL of the assay reagent, reacted in a water bath at 37 °C for 10 minutes, and measured the absorbance value at 505 nm. The result was normalized to the protein concentrations of the samples, which were determined using the BCA protein Assay Kit (Vazyme Biotech, Jiangsu, China). For the lactate assay, we utilized a lactic acid testing kit (Jiancheng Bioengineering Institute, Jiangsu, China). This kit applies NAD+ as the hydrogen acceptor. LDH can catalyze the dehydrogenation of lactic acid to produce pyruvate, which converts NAD+ into NADH. Insoluble blue–purple formazan can be produced by dehydrogenases, which originate from the interactions of nitroblue tetrazolium (NBT) and NADPH in the presence of phenazine methosulfate (PMS ). The absorbance at 530 nm is linearly related to the content of lactic acid. Then, 20 μL supernatant samples (DMEM with no phenol red) were mixed with 200 μL NBT solution and 1 mL enzyme reaction buffer. After 10 min at 37 °C, stop buffer was added, and the absorbance values (530 nm) were monitored. The lactic acid concentrations of the samples were calculated using the difference in two absorbance values at 540 nm and the lactic acid standard. The result was normalized to the protein concentrations of the samples, which were determined using the BCA protein Assay Kit (Vazyme Biotech, Jiangsu, China).
In vivo tumor formation assay
Aiming at the target lncRNA MDHDH, we designed a lentivirus expression vector using the human eukaryotic translation elongation Factor 1 α1 promoter. GL261 cells, U87 cells, C57 cells and BALB/c nude mice were chosen for the in vivo experiments. After lentivirus infection and puromycin selection, we set up the experimental cell line LV-EF1a>MDHDH-CMV>Luciferase/T2A/Puro (Cyagen, Guangzhou, China) and the control cell line LV-CMV>Luciferase/T2A/Puro (Cyagen, Guangzhou, China) for animal experiments. Four-week-old male C57 mice were purchased from Vital River Laboratories (Beijing, China). The mice were assigned randomly to two groups (n=10 each group). GL261 cells were subcutaneously injected into the right frontal lobes of the mice. The animals were anesthetized and dissected 3 weeks later. Tumor volumes were monitored by bioluminescence (IVIS Spectrum in vivo imaging system, PerkinElmer, MA, USA).
In addition, the U87 cells were injected at the same position mentioned above into three groups (n=10 each group) of four-week-old male Nu/Nu mice (Vital River Laboratories, Beijing, China). The Nu/Nu mice were anesthetized and dissected 3 weeks later.
For MDHDH overexpression with PSMA1 knockdown U87 cell line (OE MDHDH+shPSMA1), the following lentiviral expression vectors were applied: LV-U6>PSMA1-shRNA>SV40/BSDr (Origene, Jiangsu, China)
Sense sequence: GCCTGTGTCTCGTCTTGTATC
Top Strand (5'-3'): CACCGCCTGTGTCTCGTCTTGTATCTTCAAGAGAGATACAAGACGAGACACAGGC
Bottom Strand (5'-3') AAAAGCCTGTGTCTCGTCTTGTATCTCTCTTGAAGATACAAGACGAGACACAGGC
Target (3'-5'): CGGACACAGAGCAGAACATAGAAGTTCTCTCTATGTTCTGCTCTGTGTCCGAAAA
After lentivirus infection and puromycin/blasticidin double selection, the OE MDHDH+shPSMA1 U87 cell line was obtained. The same procedure as above was used for tumor formation and bioluminescence experiments.
Tumor tissues were paraformaldehyde fixed, paraffin embedded, sectioned (5 μm) and transferred onto glass slides. The deparaffinized sections were incubated in H2O2 for 10 min and rehydrated in a series of ethanol solutions. Ki67 or MDH2 staining was performed after antigen retrieval with 1 mM EDTA (pH 8), 10 mM citrate buffer, or 1 mM EDTA plus 10 mM Tris-Cl (pH 8). Sections were washed 3 times in PBS, treated with 3% H2O2 in PBS for 15 minutes, blocked in 10% goat serum and 0.3% Triton X-100 in PBS for 1 hour, and incubated with primary antibody against MDH2 or Ki67 overnight. Secondary antibody (ZSGB-BIO, Beijing, China) was applied for 30 min at 37 °C. The sections were developed with the Polink-2 plus® Polymer HRP Detection System (ZSGB-BIO, Beijing, China) according to the manufacturer’s protocol. Next, the sections were visualized by using a diaminobenzidine (DAB) substrate kit (ZSGB-BIO, Beijing, China) for 10 min. After intensive washing, the sections were counterstained with hematoxylin, dehydrated and cover-slipped. Then, the samples were observed with an Olympus BX41 microscope and an Olympus DP72 camera. Data were analyzed with FIJI software (based on ImageJ version 1.52). The intensity score was assessed as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The IHC score (histochemistry score, H-score) = (percentage of weak intensity area ×1) + (percentage of moderate intensity area ×2) + (percentage of strong intensity area ×3).
Online tools, Datasets and software
Cistrome Data browser (T98G cell line, CistromeDB:103264, Normal brain tissue, CistromeDB:6651) ;
GEPIA (Gene Expression Profiling Interactive Analysis) 1 and 2 (http://gepia.cancer-pku.cn/andhttp://gepia2.cancer-pku.cn/#index) ;
CGGA (Chinese Glioma Genome Atlas) dataset (http://www.cgga.org.cn/) ;
Metascape online tools (https://metascape.org/)  for pathway enrichment and MCODE (Molecular Complex Detection) analysis;
GTEx (Genotype-Tissue Expression) dataset;
GEO dataset: GSE60666 ; GSM772772 ; GSM3061513 ;
R software (version 1.2.1335) and the ggplot and pROC  packages.
Statistical analysis was performed using Prism 7 software (GraphPad Software, CA, USA). Student’s t test was used to compare differences between two groups. Survival curves were plotted and analyzed using the Kaplan–Meier method and log-rank test. All data were displayed as the mean ± standard error of the mean (SEM), and a P value < 0.05 was considered to be statistically significant.