Fig. 1

LncRNA MDHDH was significantly downregulated in gliomas. LncRNA profiling and public datasets reveal MDHDH is a candidate glioblastoma multiforme suppressor. A, Graphical representation of the malate–aspartate shuttle. As shown, this shuttle is operated by two pairs of enzymes, cytosolic GOT1 and MDH1 as well as mitochondrial GOT2 and MDH2, which act in concert to transfer reducing equivalents across the mitochondrial membrane. B A screening strategy was used to identify key MDH2-binding lncRNAs in GBM. RIP-seq experiments were performed to identify the top ten MDH2-binding lncRNAs. The top ten and high-throughput differentially expressed lncRNAs were intersected to obtain 4 transcripts (ENST00000623591, ENST00000452361, ENST00000598149 and MDHDH). C Box-plot analysis of MDH2-binding lncRNAs (corresponding to Figure 1B) in the microarrays (green: normal brain tissue; blue: tumor margin; red: tumor tissue). D RNA immunoprecipitation using MDH2 antibody (IgG as control) to capture RNA from glioma cell lines (U87 and U251). n=3 independent experiments, two-tailed Student’s t test. E Graphic representation of the relative MDHDH expression level (Log2(TPM+1)) in different tissues (GBM vs. GTEx, LGG vs. GTEx). GBM and LGG represent glioblastoma multiforme (GBM, WHO grade IV) and low-grade gliomas (LGG. WHO grade II and III) in TCGA datasets. GTEx represents normal brain tissue in the GTEx database (*, statistically significant). F The differential expression levels of MDHDH in the clinical glioma specimens classified into World Health Organization (WHO) grades were examined using qRT–PCR. The results are presented as the mean ± s.d. from three independent experiments. G The differential expression levels of MDHDH in the normal human astrocyte (NHA) cell line and GBM cell lines (U87 and U251). The results are presented as the mean ± s.d. from three independent experiments. H Representative RNA chromogenic in situ hybridization (CISH) images of MDHDH probe staining in human glioma tissues (WHO grade I-IV) and human normal brain tissue (upper). The CISH probe appeared brown (positive) after binding to target RNA (MDHDH). Scale bar = 100 μm. MDHDH H-score of different glioma tissues (WHO I-IV) and human normal brain tissue (right panel). I RNA fluorescence in situ hybridization (FISH) to detect Cy3-MDHDH (red) in U87 cells. Nuclei were stained with DAPI (blue), and images were merged. qRT–PCR for MDHDH expression in the cytoplasmic and nuclear RNAs isolated from NHA, U87 and U251 cells. Scale bar = 10 μm (n.s., not significant; *, P<0.05; **, P<0.01; ***, P<0.001). J Assessment of the overall survival (OS) and disease-free survival (DFS) of glioma patients with low or high LINC00632-005 (MDHDH) expression levels (cutoff value: 50%). K ROC curve analyses to evaluate the diagnostic potential of LINC00632 for GBM and LGG. L Univariate analysis of 144 glioma patients using the Cox regression model. The HR (hazard ratio) for MDHDH was 0.432 (0.251-0.746), a protective factor for the disease