Production and Stabilization of Specific Upregulated Long Noncoding RNA HOXD-AS2 in Glioblastomas Are Mediated by TFE3 and miR-661, Respectively.

Differential expression of long noncoding RNAs (lncRNA) plays a key role in the development of gliomas. Because gliomas are the most common primary central nervous system tumor and glioblastomas have poor prognosis, it is urgent to develop new diagnostic methods. We have previously reported that lncRNA HOXD-AS2, which is specifically up-regulated in gliomas, can activate cell cycle and promote the development of gliomas. It is expected to be a new marker for molecular diagnosis of gliomas, but little is known about HOXD-AS2. Here, we demonstrate that TFE3 and miR-661 maintain the high expression level of HOXD-AS2 by regulating its production and degradation. We found that TFE3 acted as a transcription factor binding to the HOXD-AS2 promoter region and raised H3K27ac to activate HOXD-AS2. As the cytoplasmic-located lncRNA, HOXD-AS2 could be degraded by miR-661. This process was inhibited in gliomas due to the low expression of miR-661. Our study explains why HOXD-AS2 was specifically up-regulated in gliomas, helps to understand the molecular characteristics of gliomas, and provids insights for the search for specific markers in gliomas.

PTB-AS, a Novel Natural Antisense Transcript, Promotes Glioma Progression by Improving PTBP1 mRNA Stability with SND1.

Glioma, the most common primary malignancy in the brain, has high recurrence and lethality rates, and thus, elucidation of the molecular mechanisms of this incurable disease is urgently needed. Poly-pyrimidine tract binding protein (PTBP1, also known as hnRNP I), an RNA-binding protein, has various mechanisms to promote gliomagenesis. However, the mechanisms regulating PTBP1 expression are unclear. Herein, we report a novel natural antisense noncoding RNA, PTB-AS, whose expression correlated positively with PTBP1 mRNA. We found that PTB-AS significantly promoted the proliferation and migration in vivo and in vitro of glioma cells. PTB-AS substantially increased the PTBP1 level by directly binding to its 3' UTR and stabilizing the mRNA. Furthermore, staphylococcal nuclease domain-containing 1 (SND1) dramatically increased the binding capacity between PTB-AS and PTBP1 mRNA. Mechanistically, PTB-AS could mask the binding site of miR-9 in the PTBP1-3' UTR; miR-9 negatively regulates PTBP1. To summarize, we revealed that PTB-AS, which maintains the PTBP1 level through extended base pairing to the PTBP1 3' UTR with the assistance of SND1, could significantly promote gliomagenesis.

Long noncoding RNA HOXD-AS2 regulates cell cycle to promote glioma progression.

Now, numerous exciting findings have been found that long noncoding RNAs (lncRNAs) play a vital role in cancer malignant progression. However, their potential involvement in glioma is not well understood. Here, we performed a high-throughput microarray to detect the lncRNA expression profiles between glioma cell lines and normal astrocyte cell lines. HOXD-AS2 was increased in glioma cells and it was associated with glioma grade and poor prognosis. Loss of HOXD-AS2 can inhibit glioma cell growth by inducing cell-cycle G1 arrest in vitro. The proliferation of glioma was inhibited followed by knocking down the expression of HOXD-AS2 not only in subcutaneous injection model but also in orthotopic implantation model. These findings indicate that HOXD-AS2 promotes the glioma progression and may serve as a potential target for glioma diagnosis and therapy.

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9.

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Targeting HOTAIRM1 ameliorates glioblastoma by disrupting mitochondrial oxidative phosphorylation and serine metabolism.

Serine hydroxymethyltransferase 2 (SHMT2), which catalyzes the conversion of serine to glycine and one-carbon transfer reactions in mitochondria, is significantly upregulated in glioblastoma (GBM). However, the mechanism by which the stability of SHMT2 gene expression is maintained to drive GBM tumorigenesis has not been clarified. Herein, through microarray screening, we identified that HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) modulates the SHMT2 level in various GBM cell lines. Serine catabolism and mitochondrial oxidative phosphorylation activities were decreased by HOTAIRM1 inhibition. Mechanistically, according to our mass spectrometry and eCLIP-seq results, HOTAIRM1 can bind to PTBP1 and IGF2BP2. Furthermore, HOTAIRM1 maintains the stability of SHMT2 by promoting the recognition of an m6A site and the interaction of PTBP1/IGF2BP2 with SHMT2 mRNA. The stability of HOTAIRM1 can also be enhanced and results in positive feedback regulation to support the progression of GBM. Thus, targeting HOTAIRM1 could be a promising metabolic therapy for GBM.

Acidosis enhances the self-renewal and mitochondrial respiration of stem cell-like glioma cells through CYP24A1-mediated reduction of vitamin D.

Acidosis is a significant feature of the tumor microenvironment in glioma, and it is closely related to multiple biological functions of cancer stem cells. Here, we found that the self-renewal ability, the mitochondrial activity and ATP production were elevated in stem cell-like glioma cells (SLCs) under acidic microenvironment, which promoted and maintained the stemness of SLCs. Under acidosis, 25-hydroxy vitamin D3-24-hydroxylase (CYP24A1) was upregulated and catalyzed the fast degradation of 1α,25(OH)2D3. We further revealed that the active form of vitamin D (1α,25(OH)2D3) could inhibit the expression of stemness markers, attenuate acidosis-induced increase of self-renewal ability and mitochondrial respiration in stem cell-like glioma cells. Our study indicates that the acidosis-CYP24A1-vitamin D pathway may be a key regulator of the cancer stem cell phenotype in malignant glioma and point out the potential value for the utilization of vitamin D to target cancer stem cells and to restrain the growth of malignant glioma in the future.

Correction: Glioma-associated human endothelial cell-derived extracellular vesicles specifically promote the tumourigenicity of glioma stem cells via CD9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glioma-associated human endothelial cell-derived extracellular vesicles specifically promote the tumourigenicity of glioma stem cells via CD9.

The perivascular niche in glioma is critical for the maintenance of glioma stem cells (GSCs), and tumour-endothelial cell (EC) communication impacts tumourigenesis in ways that are incompletely understood. Here, we show that glioma-associated human endothelial cells (GhECs), a main component of the perivascular niche, release extracellular vesicles (EVs) that increase GSC proliferation and tumour-sphere formation. GSCs treated with GhEC-EVs create a significantly greater tumour burden than do untreated GSCs in orthotopic xenografts. Mechanistic, analysis of EVs content identified CD9 as a mediator of the effects on GSCs. CD9 can activate the BMX/STAT3 signalling pathway in GSCs. Our results illuminate the tumour-supporting role of ECs by identifying that EC-derived EVs transfer of CD9 during intercellular communication, thereby enhancing the aggressiveness of glioblastoma by specifically maintaining GSCs.

RhoGDIα suppresses self-renewal and tumorigenesis of glioma stem cells.

Glioma stem cells (GSCs) are a subset of tumor cells that drive glioma initiation and progression. The molecular mechanisms underlying the maintenance of GSCs are still poorly understood. Here we investigated the role of Rho GDP dissociation inhibitor α (RhoGDIα) in GSCs. RhoGDIα was down-regulated in glioma stem cells. Over-expression of RhoGDIα suppressed the self-renewal and tumorigenesis of GSCs. Further data showed that RhoGDIα inhibited the transcription activity of stem cell marker Oct4. Moreover, inactivation of ROCK1, a downstream effector of RhoGDIα, also decreased the self-renewal and Oct4 transcription activity, and rescued the effects caused by RhoGDIα knockdown. Our results indicate that RhoGDIα is involved in the maintenance of GSCs.