The expression level of LINC01956 is elevated in GBM tissues and associated with poor clinical outcome
The Cancer Genome Atlas (TCGA) database was used to investigate the expression of lncRNAs in glioma and normal brain tissues (fold change>2, Supplementary Figure 1A). We examined the expression levels of the top thirty lncRNAs that were significantly increased in glioma tissues (Supplementary Figure 1 B) by RT-PCR. We found that the expression level of LINC01956 was elevated in glioma tissues compared with normal brain tissues (Figure 1A); therefore, we selected LINC01956 for further study. This finding was also confirmed by using the GEPIA server (http://gepia.cancer-pku.cn/, which analyzes data from TCGA and GTeX, Figure1B). In addition, GBM patients with a high expression level of LINC01956 tended to have poorer overall survival and disease-free survival rates than those with low expression levels of LINC01956(Figure1C and D, data analyzed with GEPIA). The time ROC analysis was carried out to compare the predictive accuracy and risk score of LINC01956 for OS and DFS in glioma by using the TCGA database. In the OS analysis, LINC01956 expression level could predict the prognosis of glioma patients at 1, 3 and 5 years, and its AUC under the ROC curve was 0.75, 0.808 and 0.725, respectively(Supplementary Figure 1C). In the DFS analysis, AUC under the ROC curve was 0.75, 0.805 and 0.734, respectively(Supplementary Figure 1D). These data fully implicated that LINC01956 could be an ideal marker for predication of OS and DFS in glioma.
Correlation of LINC01956 expression levels with clinicopathological features in glioma
We then analyzed the correlation of LINC01956 expression levels with clinicopathological features in glioma by mining the data from TCGA. It was found that LINC01956 expression levels increased gradually with the glioma WHO grades. Glioma patients with WHO grade 4 had the highest expression level of LINC01956 than other patients(Supplementary Figure 1E). In addition, LINC01956 expression level was highly expressed in glioblastoma patients, when compared with that in other histological types(Supplementary Figure 1F). We also found that LINC01956 expression level was decreased in IDH mutant patients when compared with that in IDH wild type patients(Supplementary Figure 1G). Interestingly, LINC01956 expression level was significantly associated with patient treatment outcome. In glioma patients with poor outcome(progressive disease), the LINC01956 expression level was significantly higher than that in those patients with clinical benefit (complete response, partial response and stable disease) (Supplementary Figure 1H). In the univariate analysis, WHO grade, 1p/19q codeletion, IDH status, histological type andLINC01956 expression level affected the prognosis of glioma patients (all p < 0.05).Furthermore, multivariate cox regression showed that the above characteristics were also independent risk factors for unfavorable OS of glioma(Table 1).
LINC01956 inhibition decreases GBM cell growth and invasion
Firstly, genes coexpressed with LINC01956 (|logFC|＞1, P.adj < 0.05) were selected to perform gene enrichment analysis, followed by GO term analysis. The biological process (BP) analysis showed positive regulation of cell cycle process, positive regulation of cell cycle phase transition, epithelial cell proliferation and positive regulation of cell−cell adhesion. The cellular component (CC) analysis showed that spindle, spindle microtubule and collagen-containing extracellular matrix were significantly enriched. The molecular fuction (MF) analysis showed that growth factor activity, chemokine activity, cytokine receptor binding and cytokine activity were significantly enriched. In the KEGG analysis, we found that cell cycle, cell adhesion molecules and focal adhesion were significantly enriched(Supplementary Figure 1I). These data implicated that LINC01956 may participate in cell signaling functions that regulate proliferation and invasion of glioma.
Based on the above findings, we then performed biological function assay to analyze LINC01956’s function on glioma cell proliferation and invasion. GBM cell lines (U87 and U251) were used in this functional study. First, we established cells in which LINC01956 was stably knocked down (sh-LINC01956) and a control cell group, namely, sh-ctrl (Figure 2A). Cell growth was examined by MTT and colony formation assays. As revealed by the MTT assay, LINC01956 downregulation decreased cell viability (Figure 2B). In parallel, the colony formation assay revealed that sh-LINC01956 cells formed smaller and fewer colonies than sh-ctrl cells(Figure 2C, Supplementary Figure 1J). We further sought to determine whether LINC01956 affects the cell cycle distribution. The frequency of GBM cells in G1 phase was significantly higher than that in S phase when LINC01956 was knocked down, as revealed by flow cytometric analysis (Figure 2D, Supplementary Figure1K). In parallel, G1/S cell cycle checkpoint proteins (e.g., cyclinD1 and CDK4) were downregulated in sh-LINC01956 cells (Figure 2E). Together, these findings imply that LINC01956 promotes the transition from G1 to S phase and thus promotes GBM cell growth in vitro. The Boyden chamber assay showed that LINC01956 inhibition decreased the invasion ability of GBM cells(Figure 2F, Supplementary Figure1L). Subsequently, we analyzed the expression levels of epithelial-mesenchymal transition (EMT)-related proteins by western blot analysis. Interestingly, LINC01956 downregulation inhibited N-cadherin and vimentin expression and elevated E-cadherin expression levels (Figure 2G). These data suggested that downregulation of LINC01956 reversed the EMT phenotype.
Subsequently, we sought to determine whether LINC01956 has an effect on cell growth in vivo. To this end, luciferase-expressing U87 cells transduced with sh-ctrl or sh-LINC01956 were injected into the brains of nude mice to establish an orthotopic glioma xenograft model. We observed that the growth of sh-LINC01956-transduced cells was significantly slower than that of sh-ctrl-transduced cells (Figure 2H). These findings indicate that downregulation of LINC01956 inhibits tumorigenesis of glioma cells in vivo.
LINC01956 is a potential target in patient-derived glioma organoid model
We established personalized patient-derived organoid (PDO) models to further investigate whether LINC01956 could be a potential target for glioma. As revealed in Figure 2I, down-regulation of LINC01956 significantly suppressed the glioma cell growth in two PDO models. Thus, our data suggest that LINC01956 could be a potential target in glioma patients.
LINC01956 promotes GBM cell progression via the WNT/β-catenin signaling pathway
We next performed RNA sequencing to examine the difference in transcript expression levels between sh-ctrl and sh-LINC01956 cells(fold change>2). Gene ontology analysis, KEGG pathway enrichment and Gene Set Enrichment Analysis(GSEA) analysis revealed that the WNT/β-catenin pathway was significantly altered when LINC01956 was downregulated (Supplementary Figure 2A-D). Western blot analysis revealed that downregulation of LINC01956 decreased the levels of phosphorylated β-catenin and its downstream targets(c-myc and cyclinD1, Figure 3A). The immunofluorescence assay revealed that downregulation of LINC01956 impaired the translocation of β-catenin into the nucleus (Figure 3B). In addition, downregulation of LINC01956 markedly decreased TOP/FOP transcriptional activity (Figure 3C). These data suggest that downregulation of LINC01956 suppresses WNT/β-catenin signaling pathway activity.
To examine whether the WNT/β-catenin pathway mediates the effect of LINC01956 on GBM cell progression, we used LiCl(a key activator of the WNT/β-catenin pathway) to perform rescue experiments in LINC01956-silenced cells. As expected, the suppressive effect of LINC01956 downregulation on cell viability and proliferation was significantly reduced in the context of LiCl addition (Figure 3 D-E). In parallel, both the cell invasion ability and cell cycle distribution, which were affected by LINC01956 downregulation, were restored after WNT/β-catenin pathway activation (Figure 3F-G).Collectively, our data suggest that the WNT/β-catenin pathway mediates the pro-motive effect of LINC01956 on GBM development.
LINC01956 binds to FUS and reduces its ubiquitination
RT-PCR showed that LINC01956 is localized in both the cytoplasm and the nucleus (Supplementary Figure 3A). The starBase database (http://starbase.sysu.edu.cn/starbase2/rbpLncRNA.php) was used to identify interactions between LINC01956 and potential RNA-binding proteins (RBPs). Among these RBPs, we found that there were three RBPs (FUS, NOP56 and TIA1) that interacted with LINC01956(Supplementary Figure3B). We focused on FUS because FUS participated in the regulation of GBM cell progression. We performed a series of experiments to confirm the association between LINC01956 and FUS. We used SDS-PAGE to isolate proteins complexes pulled down with a probe targeting LINC01956. Among the bands, that the band corresponding to FUS was present in the immune blot of the precipitate pulled down with the probe targeting LINC01956 and not in that pulled down with the control probe (Supplementary Figure3C). RNA pull-down and RIP assays revealed that LINC01956 directly interacted with FUS in U87 and U251 cells (Figure 4A). Furthermore, we used a series of LINC01956 deletion mutants to map the FUS binding region. We found that LINC01956 mutant Δ3 bound to FUS as efficiently as full-length LINC01956, whereas the binding capacity of the other mutants was completely abolished (Figure 4B). Via an immunofluorescence assay, we identified colocalization of LINC01956 and FUS in GBM cells (Figure 4C).We then demonstrated that the total protein level of FUS was decreased by LINC01956 inhibition, while its mRNA level was not altered (Figure 4D).Subsequently, a cycloheximide (CHX) chase assay was performed to examine whether LINC01956 can maintain FUS protein stability. The half-life of the FUS protein was significantly decreased to approximately 16 hours in cells with LINC01956 downregulation, whereas it was greater than 24 hours in the control group, as revealed by western blot analysis (Figure 4E). The ubiquitination assay revealed a significant increase in polyubiquitinated FUS protein in cells with LINC01956 downregulation (Figure 4F).
Taken together, these data suggest that LINC01956 binds to FUS and reduces its ubiquitination.
LINC01956 facilitates the translocation of β-catenin into the nucleus of GBM cells by recruiting FUS
The interaction between FUS and β-catenin was predicted by starBase(Supplementary Figure 3B). The co-IP assay was performed to confirm the interaction between FUS and β-catenin(Figure 5A).We then sought to determine whether FUS mediates LINC01956’s effect on the WNT/β-catenin signaling pathway. The two-step co-IP assay revealed that tagged β-catenin and FUS were both present in the anti-hemagglutinin (HA)-precipitated complex enriched with LINC01956. Similarly, HA-β-catenin and FLAG-FUS coprecipitated with LINC01956-FLAG but not with immunoglobulin G (IgG) (Figure 5B). These data suggest that β-catenin, FUS and LINC01956 form a complex. In addition, we observed that overexpression of FUS counteracted the decrease in the level of nuclear phosphorylated (p)β-catenin in sh-LINC01956 GBM cells (Figure 5C). In parallel, the immunofluorescence assay revealed that downregulation of LINC01956 inhibited nuclear translocation of β-catenin and that this effect was abrogated in the context of FUS cotransfection (Figure 5D). Taken together, we speculate that LINC01956 leads to nuclear translocation of activated β-catenin in cooperation with FUS.
Exosomal LINC01956 promotes M2 polarization of macrophages
The tumor-infiltrating lymphocytes may affect prognosis of various of cancer, including glioma. We then explored the association between LINC01956 and 24 immune-cell subsets in glioma by means of ssGSEA with Spearman r. The data were present in supplementary Figure 2D. Among the analysis, the significant correlation between LINC01956 and infiltration levels of macrophages caught our attention. Because activation of the Wnt/β-catenin pathway contributes to M2 polarization; we thus sought to determine whether LINC01956 affectsM2 polarization. To this end, we evaluated the expression levels of LINC01956, M1 markers, and M2 markers in unpolarized macrophages, LPS/INF-γ-induced M1 macrophages, and IL-4/IL-13-induced M2 macrophages. We observed elevated expression of M1-associated genes (CD80, MCP-1 and iNOS) in M1 macrophages and of M2-associated genes (CD206 and MRC-2) in M2 macrophages(Figure 6A).This result indicates the successful polarization of macrophages. Compared with that in M1 macrophages, the expression level of LINC01956 in M2 macrophages was elevated (Figure 6B). These data suggest that LINC01956 is involved in macrophage polarization. Subsequently, we treated THP-1 cells with PMA for 24 hours, transfected them with si-ctrl or si-LINC01956 and added IL-4 and IL-13 to induce polarization toward the M2 phenotype. In si-LINC01956 cells, the levels of M1 markers were markedly increased, and the levels of M2markers were significantly decreased (Figure 6C). In contrast, LINC01956 overexpression led to the opposite result(Figure 6D). In addition, when compared with supernatant from control cells, supernatant from pcDNA-LINC01956 cells led to increased expression of M2 markers (Figure 6E).
Subsequently, we explored the interactions between GBM cells and macrophages. Previous studies demonstrated that lncRNAs can be transferred by exosomes and thereby regulate the tumor microenvironment (TME). We hypothesized that LINC01956 may be transferred in this way. We isolated exosomes from the supernatants of cultured GBM cells and measured the levels of the exosome-related proteins CD63, HSP70, and HSP90 by western blot analysis (Figure 6F). Downregulation of LINC01956 led to decreased levels of LINC01956 in the secreted exosomes (Figure 6G). These data proved the existence of LINC01956 in exosomes. Subsequently, we cocultured unpolarized macrophages with exosomes isolated from pcDNA-LINC01956 control cells. The expression levels of the M2 phenotype markers CD206 and MRC-2 were elevated in the pcDNA-LINC01956 group but not in the control group (Figure 6H). This finding indicates that exosomal LINC01956 promotes M2 polarization.
Collectively, our findings suggest that LINC01956 can be transferred via exosomes, thereby promoting M2 polarization of macrophages.
LINC01956 was transcriptionally regulated by HIF-1α under hypoxic conditions
Previous studies have demonstrated that a hypoxic TME might contribute to abnormal expression of some lncRNAs, including in GBM. We sought to determine whether LINC01956 is a hypoxia-sensitive lncRNA. We exposed GBM cells to hypoxia or normoxia for 48 h and revealed that HIF-1α and LINC01956 expression levels were elevated (Figure 7A and B) under hypoxic conditions. The efficiency of HIF-1α downregulation was examined by using western blotting (Figure 7C). Downregulation of HIF-1α significantly inhibited LINC01956 expression under both normoxic and hypoxic conditions. In addition, downregulation of HIF-1α counteracted hypoxia-induced LINC01956 upregulation(Figure 7D). We then explored whether HIF-1αregulates LINC01956 by binding to its promoter. Via UCSC and JASPAR bioinformatics software, we analyzed the 1-kb region upstream of the transcription start site of LINC01956 and identified a putative HIF-1α response element (HRE) in the LINC01956 promoter region (positions -367 to -372) (Figure 7E). To understand whether HIF-1α regulates LINC01956 expression via this HRE, a vector carrying the wild-type LINC01956 promoter and a vector carrying the mutant LINC01956 promoter were constructed. There was an increase in luciferase activity in cells cotransfected with the pcDNA3-HIF-1α plasmid and wild-type LINC01956 promoter. Luciferase activity was impaired in cells that were cotransfected with the pcDNA3-HIF-1α plasmid and mutant LINC01956 promoter (Figure 7F). Subsequently, the ChIP assay was carried out to confirm that HIF-1α directly bound to the LINC01956 promoter (Figure 7G). Taken together, our findings imply that LINC01956 is regulated by HIF-1α.