DNA-methylation-mediated activating of lncRNA SNHG12 promotes temozolomide resistance in glioblastoma.

BACKGROUND: Accumulating evidence shows that long noncoding RNAs (lncRNAs) are important regulator molecules involved in diverse biological processes. Acquired drug resistance is a major challenge in the clinical treatment of glioblastoma (GBM), and lncRNAs have been shown to play a role in chemotherapy resistance. However, the underlying mechanisms by which lncRNA mediates TMZ resistance in GBM remain poorly characterized. METHODS: Quantitative reverse transcription PCR (qRT-PCR) and fluorescence in situ hybridization assays were used to detect small nucleolar RNA host gene 12 (SNHG12) levels in TMZ-sensitive and TMZ-resistant GBM cells and tissues. The effects of SNHG12 on TMZ resistance were investigated through in vitro assays (western blots, colony formation assays, flow cytometry assays, and TUNEL assays). The mechanism mediating the high expression of SNHG12 in TMZ-resistant cells and its relationships with miR-129-5p, mitogen-activated protein kinase 1 (MAPK1), and E2F transcription factor 7 (E2F7) were determined by bioinformatic analysis, bisulfite amplicon sequencing, methylation-specific PCR, dual luciferase reporter assays, chromatin immunoprecipitation assays, RNA immunoprecipitation assays, immunofluorescence, qRT-PCR, and western blot. For in vivo experiments, an intracranial xenograft tumor mouse model was used to investigate SNHG12 function. RESULTS: SNHG12 was upregulated in TMZ-resistant cells and tissues. Overexpression of SNHG12 led to the development of acquired TMZ resistance, while knockdown of SNHG12 restored TMZ sensitivity. An abnormally low level of DNA methylation was detected within the promoter region of SNHG12, and loss of DNA methylation made this region more accessible to the Sp1 transcription factor (SP1); this indicated that methylation and SP1 work together to regulate SNHG12 expression. In the cytoplasm, SNHG12 served as a sponge for miR-129-5p, leading to upregulation of MAPK1 and E2F7 and endowing the GBM cells with TMZ resistance. Disinhibition of MAPK1 regulated TMZ-induced cell apoptosis and the G1/S cell cycle transition by activating the MAPK/ERK pathway, while E2F7 dysregulation was primarily associated with G1/S cell cycle transition. Clinically, SNHG12 overexpression was associated with poor survival of GBM patients undergoing TMZ treatment. CONCLUSION: Our results suggest that SNHG12 could serve as a promising therapeutic target to surmount TMZ resistance, thereby improving the clinical efficacy of TMZ chemotherapy.

LncRNA-mRNA Co-expression Profiles Relative to Vascular Remodeling in Moyamoya Patients Without RNF213 Mutation.

OBJECTIVE: Moyamoya disease (MMD) is an idiopathic cerebrovascular disease with unknown etiology. Long noncoding RNA (lncRNA) and messenger RNA (mRNA) profiles in MMD remain unknown. In this current study, we aim to investigate lncRNA-mRNA co-expression pattern and their biological functions in superficial temporal artery (STA) of MMD. METHODS: STA of 3 MMD patients without RNF213 mutation and 3 age-matched controls were obtained for transcriptomic RNA sequencing. Bioinformatics analysis was performed to investigate their molecular functions and interactions. Then, differentially expressed genes relative to vascular remodeling were further validated by quantitative real-time polymerase chain reaction and immunofluorescence. WNT5A functions were tested by tube formation assay and wound scratching assay in human microvascular endothelial cells (HMECs). RESULTS: We detected 6235 different lncRNAs and 2065 different mRNAs from the RNA-sequencing between MMD patients and controls (P < 0.05; fold change >2.0). Gene ontology showed that altered mRNAs were enriched for endothelial cell morphogenesis and positive regulation of angiogenesis, which were closely related with vascular remodeling. We then searched 76 altered genes related with vascular remodeling and applied Kyoto Encyclopedia of Genes and Genomes analysis. Integrated analysis of lncRNA-TF-mRNA co-expression networks and gene verifications indicated that molecular including WNT5A, TEK, and GATA2 may contribute to the vascular malformation of MMD. Overexpression of WNT5A in HMECs promoted tube formation and cell migration. CONCLUSIONS: In MMD patients, genes related to vascular remodeling including WNT5A and their regulators were aberrantly disrupted. These results will help elucidate the complicated pathogenic mechanism of MMD and develop potential therapeutic targets facilitating MMD angiogenesis in the future.

Caveolin-1 Promoted Collateral Vessel Formation in Patients With Moyamoya Disease.

Background: Caveolin-1 (Cav-1) plays pivotal roles in the endothelial function and angiogenesis postischemia. Moyamoya disease (MMD) is characterized by progressive artery stenosis with unknown etiology. We aim to determine whether serum Cav-1 levels of patients with MMD were associated with collateral vessel formation after bypass surgery. Methods: We studied serum Cav-1 levels of 130 patients with MMD (16 with RNF213 p.R4810K mutation and 114 without RNF213 p.R4810K mutation), 15 patients with acute stroke, and 33 healthy controls. Cerebral perfusion and collateral circulation were evaluated preoperation and at 6 months after operation using pseudocontinuous arterial spin labeling MRI (pCASL-MRI) and digital subtraction angiography (DSA), respectively. Endothelial expression of Cav-1 was verified in the superficial temporal artery (STA) wall of patients with MMD by immunofluorescence double staining. We also investigated whether overexpression of Cav-1 affects cell migration and tube formation using human microvascular endothelial cells (HMECs). Results: The serum Cav-1 level of patients with MMD intermediated between the stroke group and healthy controls and it was enhanced after the bypass surgery (681.87 ± 311.63 vs. 832.91 ± 464.41 pg/ml, p = 0.049). By 6 months after bypass surgery, patients with MMD with better collateral compensation manifested higher postoperative/preoperative Cav-1 ratio (rCav-1) than bad compensation patients. Consistently, cerebral blood flow (CBF) determined by pCASL-MRI (nCBFMCA ratio) was positively in line with rCav-1 ratio (r = 0.8615, p < 0.0001). Cav-1 was expressed in the endothelial cells of the STA vessels of patients with MMD. Overexpression of Cav-1 by plasmid transfection in HMECs promoted tube formation and cell migration. Conclusion: This study indicated that Cav-1 may be a potential driver to promote angiogenesis and collateral formation after bypass surgery in patients with MMD, providing a better understanding of MMD pathophysiology and potential non-surgical targets of MMD.

Baseline Hemodynamic Impairment and Revascularization Outcome in Newly Diagnosed Adult Moyamoya Disease Determined by Pseudocontinuous Arterial Spin Labeling.

OBJECTIVE: The study aimed to investigate the hemodynamic features and independent predictors of neoangiogenesis after revascularization in moyamoya disease (MMD) by pseudocontinuous arterial spin labeling magnetic resonance imaging (pCASL MRI). METHODS: Thirty-nine MMD patients were categorized into infarction group, hemorrhagic group, and atypical group. All patients underwent combined bypass surgery and pCASL MRI with postlabeling delays (PLD) of 1525 ms and 2525 ms. Absolute CBFMCA (cerebral blood flow in middle cerebral artery territory), relative CBFMCA (CBFMCA 2525 ms/CBFMCA 1525 ms), and spatial coefficient of variation of MCA (CoVMCA) were analyzed. Relationships between CBFMCA and the following clinical parameters were assessed: Suzuki stage, modified Rankin scale (mRS), cerebrovascular accident lesion score, and deep medullary veins score. Potential predictors for favorable neoangiogenesis and hemodynamic changes were explored as well. RESULTS: Preoperative CBFMCA differed among MMD patients with variable clinical presentations, Matsushima stages, modified Rankin Scale scores, CVA scores, and deep medullary vein scores. After bypass surgery, mean CBFMCA increased significantly in the infarction group (P = 0.027) and decreased in the hemorrhagic group (P = 0.043), while spatial CoVMCA was observed to decline in all groups. Higher preoperative relative CBFMCA and spatial CoVMCA were independent predictors for robust neoangiogenesis after bypass. The cutoff value of 0.330 of spatial CoVMCA at long PLD yielded the best sensitivity at 82.1% and specificity at 81.8%. Furthermore, both preoperative relative CBFMCA and spatial CoVMCA showed mild positive correlations with ΔmRS in MMD patients. CONCLUSIONS: pCASL-MRI with multiple PLDs could reflect preoperative hemodynamic impairment and predict the neoangiogenesis after combined bypass surgery in moyamoya patients.

Association between SNAP25 and human glioblastoma multiform: a comprehensive bioinformatic analysis.

BACKGROUND: Glioblastoma multiforme (GBM) is a most common aggressive malignant brain tumor. In recent years, targeted therapy has been increasingly applied in GBM treatment. METHODS: In the present study, GSE22866 was downloaded from gene expression omnibus (GEO). The genomic and clinical data were obtained from TCGA. The differentially expressed genes (DEGs) were identified and functional analysis was performed using clusterprofiler. Then, the co-expression network for the DEGs was established using the "WGCNA" package. Next, the protein-protein interaction (PPI) was assessed using Search Tool for the Retrieval of Interacting Genes Database (STRING) and hub modules in Cytoscape were screened. The Venn diagram was plotted to showcase the overlapped hub DEGs in PPI network and TCGA. Univariate and multivariate Cox proportional hazards regression analyses were performed to predict the risk score of each patient. Validations of the hub gene were completed in other databases. RESULTS: Functional analysis of the DEGs verified the involvement of DEGs in growth factor binding and gated channel activity. Among the 10 GBM-related modules, the red one displayed the strongest tie with GBM. VAMP2 was filtered out as the most intimate protein. The PPI network and TCGA were comprehensively analyzed. Finally, SNAP25 was identified as a real hub gene positively correlated with GBM prognosis. The result was validated by GEPIA, ONCOMINE database and qRT-PCR. CONCLUSIONS: SNAP25 might act as a GBM suppressor and a biomarker in GBM treatment.