Effects of SNAP25 and VAMP2 on biological behaviors and their mechanisms in glioblastoma
10.3760/cma.j.cn115354-20250611-00337
- VernacularTitle:SNAP25与 VAMP2对胶质母细胞瘤生物学行为的影响及其机制研究
- Author:
Haichun LI
1
;
Zirui HUANG
;
Rongjun QIAN
Author Information
1. 河南省人民医院神经外科，郑州 450003
- Publication Type:Journal Article
- Keywords:
Glioblastoma;
Hub gene;
Synaptosomal-associated protein 25;
Vesicle-associated membrane protein 2;
Weighted gene co-expression network analysis;
Gene set e
- From:
Chinese Journal of Neuromedicine
2025;24(8):767-779
- CountryChina
- Language:Chinese
-
Abstract:
Objective:Glioblastoma (GBM) is a primary malignant tumor in the central nervous system, whose tissue heterogeneity and invasive growth characteristics lead to a very poor prognosis for patients. Hub genes in GBM are screened by bioinformatics analysis; expressions of Hub genes in human GBM tissue, and their effects on GBM biological behaviors and Notch signaling pathway proteins are explored, and their regulatory roles in GBM proliferation in xenograft tumor model mice are evaluated.Methods:(1) The gene expression profiles of GBM tissue and normal brain tissue from the Gene Expression Omnibus (GEO) database were obtained; differentially expressed genes (DEGs) were screened, and gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were conducted on DEGs. A co-expression network was constructed using weighted gene co-expression network analysis (WGCNA) to identify modules significantly related to GBM. Gene set enrichment analysis (GSEA) on the related module genes was performed; STRING database (version 12.0) was used to construct protein-protein interaction (PPI) network and screen the hub genes. (2) Normal brain tissue samples from 8 patients with epilepsy and GBM tissue samples from 10 patients with GBM who underwent surgical resection in Department of Neurosurgery, He'nan Provincial People's Hospital from January 1, 2022 to December 1, 2023 were collected; expressions of synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) were detected by immunohistochemical staining and Western blotting. (3) LN229 and U87 cells were routinely cultured in vitro and divided into shRNA-SNAP25/shRNA-VAMP2 group and empty vector group, and then, cells in each group were transfected with shRNA-SNAP25/shRNA-VAMP2 lentivirus or empty vector lentivirus, respectively; 24 hours after transfection, SNAP25 and VAMP2 mRNA and protein expressions in the 2 groups were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting. LN229 and U87 cells were routinely cultured in vitro and divided into shRNA-SNAP25 group, shRNA-VAMP2 group and empty vector group; cells in each group were transfected with shRNA-SNAP25 lentivirus, shRNA-VAMP2 lentivirus or empty vector lentivirus, respectively; 48 hours after transfection, proliferation was detected by clone formation assay, proliferating cell nuclear antigen Ki-67 expression was detected by immunofluorescent staining, invasion was detected by Transwell invasion assay, and Notch1, HEY1 and HES1 protein expressions in Notch signaling pathway of LN229 cells were detected by Western blotting. (4) LN229 cells transfected with shRNA-SNAP25 lentivirus, shRNA-VAMP2 lentivirus or empty vector lentivirus for 48 hours were subcutaneously injected into the right axilla of 4-week-old BALB/c nude mice, respectively, as shRNA-SNAP25 transplantation group, shRNA-VAMP2 transplantation group and empty vector transplantation group (5 mice in each group); on 28 th day of injection, immunohistochemical staining was used to detect the Ki-67 expression in the LN229 cell-transplanted tumor tissues. Results:(1) A total of 1,473 DEGs were screened, of which 880 were upregulated and 593 were downregulated. WGCNA indicated that DEGs were divided into 5 modules (greenish-blue, blue, black, brown and gray ones), among which the greenish-blue module was significantly negatively correlated with GBM ( r=-0.700, P<0.001); GSEA analysis showed that the greenish-blue module mainly involved Notch signaling pathway, and PPI network analysis identified SNAP25 and VAMP2 as hub genes. (2) Immunohistochemical staining results showed that expressions of SNAP25 and VAMP2 in GBM tissue were significantly lower than those in normal brain tissue ( P<0.05). (3) Compared with those in the empty vector group, the SNAP25 mRNA and protein expressions in LN229 and U87 cells of the shRNA-SNAP25 group were statistically decreased ( P<0.05). Compared with those in the empty vector group, the VAMP2 mRNA and protein expressions in LN229 and U87 cells of the shRNA-VAMP2 group were significantly decreased ( P<0.05). Compared with the empty vector group, the LN229 and U87 cells in the shRNA-SNAP25 group and shRNA-VAMP2 group had significantly increased colony formation number, Ki-67 expression and invasive cell number ( P<0.05). Compared with the empty vector group, LN229 cells in the shRNA-SNAP25 group and shRNA-VAMP2 group had statistically increased Notch1, HEY1, and HES1 protein expressions ( P<0.05). Immunohistochemical staining results showed that compared with that in the empty vector group (1.00±0.00), the Ki-67 expression in LN229 cell-transplanted tumor tissues of the shRNA-SNAP25 group and shRNA-VAMP2 group was statistically increased (1.41±0.05, 1.40±0.09, P<0.05). Conclusion:Hub genes SNAP25 and VAMP2 may negatively regulate the malignant biological behavior of GBM through Notch pathway, which might be the new candidate targets for GBM precise treatment.
