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Mounting evidence reveals that dysregulation of circular RNAs (circRNAs) is involved in the development of glioblastoma. Leucine-rich repeat-containing 4 (LRRC4) has been shown to suppress tumors in glioblastoma. However, whether LRRC4 can regulate the formation of circRNA is not yet understood. In this study, LRRC4 was found to interact with SAM68. LRRC4 promoted the generation of circCD44 by inhibiting the binding between SAM68 and CD44 pre-mRNA. Moreover, downregulated expression of circCD44 was found in glioblastoma multiforme (GBM) tissues and GBM primary cells. Re-expression of circCD44 significantly suppressed the proliferation, colony formation, and invasion of GBM cells and inhibited tumor growth in vivo. Mechanistically, circCD44 could regulate the expression of SMAD6 via sponging miR-326 and miR-330-5p involved in the progression of GBM. Thus, the LRRC4/SAM68/circCD44/miR-326/miR-330-5p/SMAD6 signaling axis could be a potential target for GBM treatment.

Abstract Background Leucine rich repeat containing 4 (LRRC4), also known as netrin-G ligand-2 (NGL-2), belongs to the superfamily of LRR proteins and serves as a receptor for netrin-G2. LRRC4 regulates the formation of excitatory synapses and promotes axon differentiation. Mutations in LRRC4 occur in Autism Spectrum Disorder (ASD) and intellectual disability. Multiple sclerosis (MS) is a chronic neuroinflammatory disease with spinal cords demyelination and neurodegeneration. Here, we sought to investigate whether LRRC4 is involved in spinal cords neuron-associated diseases. Methods LRRC4 was detected in the CNS of experimental autoimmune encephalomyelitis (EAE) mice by the use of real-time PCR and western blotting. LRRC4−/− mice were created and immunized with myelin oligodendrocyte glycoprotein peptide (MOG)35–55. Pathological changes in spinal cords of LRRC4−/− and WT mice 15 days after immunization were examined by using hematoxylin and eosin (H&E), Luxol Fast Blue (LFB) staining and immunohistochemistry. The number of Th1/Th2/Th17/Treg cells in spleens and blood were measured with flow cytometry. Differential gene expression in the spinal cords from WT and LRRC4−/− mice was analyzed by using RNA sequencing (RNA-seq). Adeno-associated virus (AAV) vectors were used to overexpress LRRC4 (AAV-LRRC4) and were injected into EAE mice to assess the therapeutic effect of AAV-LRRC4 ectopic expression on EAE. Results We report that LRRC4 is mainly expressed in neuron of spinal cords, and is decreased in the spinal cords of the EAE mice. Knockout of LRRC4 have a disease progression quickened and exacerbated with more severe myelin degeneration and infiltration of leukocytes into the spinal cords. We also first found that Rab7b is high expressed in EAE mice, and the deficiency of LRRC4 induces the elevated NF-κB p65 by up-regulating Rab7b, and up-regulation of IL-6, IFN-γ and down-regulation of TNF-α, results in more severe Th1 immune response in LRRC4−/− mice. Ectopic expression of LRRC4 alleviates the clinical symptoms of EAE mice and protects the neurons from immune damages. Conclusions We identified a neuroprotective role of LRRC4 in the progression of EAE, which may be used as a potential target for auxiliary support therapeutic treatment of MS.

Long non-coding RNAs (lncRNAs) play a significant role in post-translational modifications of proteins, yet the importance of lncRNAs for SUMOylation is unknown. rhabdomyosarcoma 2 associated transcript (RMST) expression in glioma tissues and normal brain tissues was measured by quantitative real-time PCR and in situ hybridization. The functional roles of RMST in astrocytomas were demonstrated by a series of in vitro experiments. The potential mechanisms of RMST for SUMOylation were investigated by RNA immunoprecipitation, RNA pull-down, western blotting, and coimmunoprecipitation assays. We first demonstrated the oncogenic activity of lncRNA RMST by inhibiting glioma cells mitophagy. We also first determined that RMST is an enhancer of FUS SUMOylation, especially boosting SUMO1 modification at K333. SUMOylation induced by RMST contributes to the interaction between FUS and heterogeneous nuclear ribonucleoprotein D (hnRNPD) and stabilized their expression and cells mitophagy. Importantly, lncRNA RMST could serve as a promising prognostic factor for glioma patients. Our results demonstrated a previously unknown function of lncRNAs worked as an enhancer in FUS SUMOylation, and RMST will be a significant guide for the development of medications targeting gliomas. Keywords: lncRNA, SUMOylation, oncogene, GBM

PELATON is a long noncoding RNA also known as long intergenic nonprotein coding RNA 1272 (LINC01272). The known reports showed that PELATON functions as an onco-lncRNA or a suppressor lncRNA by suppressing miRNA in colorectal cancer, gastric cancer and lung cancer. In this study, we first found that PELATON, as an onco-lncRNA, alleviates the ferroptosis driven by mutant p53 and promotes mutant p53-mediated GBM proliferation. We also first confirmed that PELATON is a new ferroptosis suppressor lncRNA that functions as a ferroptosis inhibitor mainly by mutant P53 mediating the ROS ferroptosis pathway, which inhibits the production of ROS, reduces the levels of divalent iron ions, promotes the expression of SLC7A11, and inhibits the expression of ACSL4 and COX2.PELATON can inhibit the expression of p53 in p53 wild-type GBM cells and regulate the expression of BACH1 and CD44, but it has no effect on p53, BACH1 and CD44 in p53 mutant GBM cells. PELATON and p53 can form a complex through the RNA binding protein EIF4A3. Knockdown of PELATON resulted in smaller mitochondria, increased mitochondrial membrane density, and enhanced sensitivity to ferroptosis inducers to inhibit GBM cell proliferation and invasion. In addition, we established a favourite prognostic model with NCOA4 and PELATON. PELATON is a promising target for the prognosis and treatment of GBM.

Abstract Background Despite the overwhelming number of investigations on AKT, little is known about lncRNA on AKT regulation, especially in GBM cells. Methods RNA-binding protein immunoprecipitation assay (RIP) and RNA pulldown were used to confirm the binding of LINC00470 and fused in sarcoma (FUS). Confocal imaging, co-immunoprecipitation (Co-IP) and GST pulldown assays were used to detect the interaction between FUS and AKT. EdU assay, CCK-8 assay, and intracranial xenograft assays were performed to demonstrate the effect of LINC00470 on the malignant phenotype of GBM cells. RT-qPCR and Western blotting were performed to test the effect of LINC00470 on AKT and pAKT. Results In this study, we demonstrated that LINC00470 was a positive regulator for AKT activation in GBM. LINC00470 bound to FUS and AKT to form a ternary complex, anchoring FUS in the cytoplasm to increase AKT activity. Higher pAKT activated by LINC00470 inhibited ubiquitination of HK1, which affected glycolysis, and inhibited cell autophagy. Furthermore, higher LINC00470 expression was associated with GBM tumorigenesis and poor patient prognosis. Conclusions Our findings revealed a noncanonical AKT activation signaling pathway, i.e., LINC00470 directly interacts with FUS, serving as an AKT activator to promote GBM progression. LINC00470 has an important referential significance to evaluate the prognosis of patients.

Abstract Background SIX homeobox 3 (SIX3) is a member of the sine oculis homeobox transcription factor family. It plays a vital role in the nervous system development. Our previous study showed that the SIX3 gene is hypermethylated, and its expression is decreased in astrocytoma, but the role of SIX3 remains unknown. Methods Chromatin-immunoprecipitation (ChIP) and luciferase reporter assay were used to confirm the binding of SIX3 to the promoter regions of aurora kinase A (AURKA) and aurora kinase B (AURKB). Confocal imaging and co-immunoprecipitation (Co-IP) were used to detect the interaction between AURKA and AURKB. Flow cytometry was performed to assess the effect of SIX3 on cell cycle distribution. Colony formation, EdU incorporation, transwell, and intracranial xenograft assays were performed to demonstrate the effect of SIX3 on the malignant phenotype of astrocytoma cells. Results SIX3 is identified as a novel negative transcriptional regulator of AURKA and AURKB, and it decreases the expression of AURKA and AURKB in a dose-dependent manner in astrocytoma cells. Importantly, interactions between AURKA and AURKB stabilize and protect AURKA/B from degradation, and overexpression of SIX3 does not affect these interactions; SIX3 also acts as a tumor suppressor, and it increases p53 activity and expression at the post-translational level by the negative regulation of AURKA or AURKB, reduces the events of numerical centrosomal aberrations and misaligned chromosomes, and significantly inhibits the proliferation, invasion, and tumorigenesis of astrocytoma in vitro and in vivo. Moreover, experiments using primary cultured astrocytoma cells indicate that astrocytoma patients with a low expression of SIX3 and mutant p53 are more sensitive to treatment with aurora kinase inhibitors. Conclusion SIX3 is a novel negative transcriptional regulator and acts as a tumor suppressor that directly represses the transcription of AURKA and AURKB in astrocytoma. For the first time, the functional interaction of AURKA and AURKB has been found, which aids in the protection of their stability, and partially explains their constant high expression and activity in cancers. SIX3 is a potential biomarker that could be used to predict the response of astrocytoma patients to aurora kinase inhibitors.

Tumor-associated macrophages (TAMs) constitute a major component of inflammatory cells in the glioblastoma multiforme (GBM) tumor microenvironment. TAMs have been implicated in GBM angiogenesis, invasion, local tumor recurrence, and immunosuppression. Coagulation factor X (FX) is a vitamin K-dependent plasma protein that plays a role in the regulation of blood coagulation. In this study, we first found that FX was highly expressed and positively correlated with TAM density in human GBM. FX exhibited a potent chemotactic capacity to recruit macrophages and promoted macrophages toward M2 subtype polarization, accelerating GBM growth. FX bound to extracellular signal-related kinase (ERK)1/2 and inhibited p-ERK1/2 in GBM cells. FX was secreted in the tumor microenvironment and increased the phosphorylation and activation of ERK1/2 and AKT in macrophages, which may have been responsible for the M2 subtype macrophage polarization. Moreover, although the lncRNA CASC2c has been verified to function as a miR-101 competing endogenous RNA (ceRNA) to promote miR-101 target genes in GBM cells, we first confirmed that CASC2c did not function as a miR-338-3p ceRNA to promote FX expression, and that FX was a target gene of miR-338-3p. CASC2c interacted with and reciprocally repressed miR-338-3p. Both CASC2c and miR-388-3p bound to FX and commonly inhibited its expression and secretion. CASC2c repressed M2 subtype macrophage polarization. Taken together, our findings revealed a novel mechanism highlighting CASC2c and FX as potential therapeutic targets to improve GBM patients by altering the GBM microenvironment.