To study the expression characteristics of CLSPN in glioma, the influence of CLSPN on the biological function of glioma and the molecular signaling pathway.

The expression characteristics of CLSPN in nervous system and its relationship with patient survival time were investigated by using human protein database [The Cancer Genome (TCGA) and the glioma database Chinese Glioma Genome Atlas (CGGA)]. The level of protein expression of CLSPN in glioma and its relationship to patient survival time were analyzed by immunohistochemical staining experiments ofCLSPN via glioma specimen chip. shRNA was used to interfere the expression of CLSPN in U87 MG cells, and then the effects of CLSPN on the proliferation and cycle of U87 MG cells were analyzed by CCK8 cell proliferation assay, EdU staining, cell monoclonal antibody assay and cell cycle assay. The molecular signaling pathways involved in CLSPN were analyzed by Genecards data, and the effect of interfering CLSPN protein expression on the expression of key proteins in cell cycle was verified by western blotting.

Human gene and protein database data showed that the transcription level of CLSPN in central nervous system tissues was low. CLSPN transcription was higher in glioma cell lines. TCGA combined with GTEx data analysis found that compared with normal brain tissue, the transcription level of high-grade glioma CLSPN was significantly higher (P<0.05), while there was no significant difference in low grade glioma (P>0.05); Moreover, there were significant differences in median survival time between glioma patients with high and low CLSPN transcription levels (P<0.05), the higher the transcription level of CLSPN, the shorter the median survival time of glioma patients. Tissue microarray results showed that CLSPN protein levels in glioblastoma were significantly higher than those in normal brain tissue (P<0.05). Compared with the no-load plasmid group, the reduction of CLSPN expression in U87 MG cells resulted in a decrease of the proliferation of U87 MG cells, the number of EdU positive cells, and the number of clonal groups, the difference was statistically significant (P<0.05). The number of G1/G0 cells in U87 MG cells increased after Interference with CLSPN, and the difference was statistically significant (P<0.05). TCGA transcriptional data show that transcription levels of CLSPN were significantly positively associated with Ki67 (MKI67) transcription levels (P<0.05). In vivo tumor formation experiments showed that the growth of U87 MG in the brain of nude mice was significantly inhibited after CLSPN expression was knocked out, and there were statistically significant differences in tumor signal values between the no-load plasmid group and the group that interfered with CLSPN expression (P<0.05), and there were statistically significant differences in survival curves between the no-load plasmid group and the CLSPN expression interference group (P<0.05). The survival time of mice in the CLSPN expression interference group was prolonged. Enrichment analysis of signaling pathways showed that the top 10 molecular signaling pathways involved by CLSPN included cell cycle checkpoint pathways. Protein imprinting detection showed that the protein expression levels of P53, p21 and p27 were significantly increased after knockdown of CLSPN in U87 MG cells, while the protein expression levels of CDK2, CDK4, Cyclin D and Cyclin E1 were significantly decreased.

CLSPN is highly expressed in glioma, and a high level of CLSPN predicts a poor survival in glioma patients. CLSPN regulates the cell G0/G1 phase by regulating the P53-p21/p27 molecular signaling pathway, thus affecting the proliferation ability of glioblastoma. CLSPN may be a potential therapeutic target for glioblastoma.