CLSPN在胶质瘤中的表达及生物学功能

Expression and biological function of CLSPN in glioma

目的研究CLSPN在胶质瘤中的表达特征,以及CLSPN对胶质瘤肿瘤生物学功能的影响及分子信号通路。方法通过人类蛋白数据库(TCGA)和胶质瘤数据库(CGGA)探讨CLSPN在胶质瘤中的转录水平特征,及其与患者生存期的关系;通过胶质瘤标本芯片进行CLSPN的免疫组织化学染色实验分析CLSPN在胶质瘤中蛋白表达水平,及其与患者生存期的关系。用shRNA干扰CLSPN在U87 MG细胞中的表达,然后通过CCK8细胞增殖检测、EdU染色、细胞单克隆实验、细胞周期实验分析CLSPN对U87 MG细胞增殖和周期的影响;通过Genecards数据分析CLSPN参与的分子信号通路,通过蛋白印记实验验证干扰CLSPN蛋白表达对细胞周期关键蛋白表达量的影响。结果人基因和蛋白库数据显示CLSPN在中枢神经系统组织中的转录水平较低;CLSPN在胶质瘤细胞系中的转录水平较高。TCGA联合GTEx数据分析发现,相比较于正常脑组织,高级别胶质瘤CLSPN的转录水平显著增高(P<0.05),而低级别胶质瘤则差异无统计学意义(P>0.05);并且,高CLSPN转录水平和低CLSPN转录水平的胶质瘤患者中位生存期差异有统计学意义(P<0.05),CLSPN的转录水平越高,胶质瘤患者的中位生存期越短。组织芯片结果显示,CLSPN在胶质母细胞瘤中的蛋白水平显著高于正常脑组织(P<0.05)。相比较于空载质粒组,干扰U87 MG细胞CLSPN表达后,U87 MG细胞增殖减慢、EdU阳性细胞数减少、形成克隆团数减少,差异均有统计学意义(P<0.05)。干扰CLSPN后U87 MG细胞G1/G0期细胞数量增加,差异有统计学意义(P<0.05)。TCGA转录组数据显示,CLSPN转录水平与Ki67(MKI67)转录水平成显著正相关(P<0.05)。体内成瘤实验发现,敲除CLSPN表达后U87 MG在裸鼠颅内生长明显抑制,空载质粒组和干扰CLSPN表达组肿瘤信号值差异有统计学意义(P<0.05),并且,空载质粒组和干扰CLSPN表达组间生存曲线差异有统计学意义(P<0.05),干扰CLSPN表达组小鼠生存期延长。信号通路富集分析可见,CLSPN参与的分子信号通路前10位包括细胞周期检查点通路。蛋白印记检测发现,敲低U87 MG细胞CLSPN的表达后,P53、p21、p27的蛋白表达量明显升高,而CDK2、CDK4、Cyclin D、Cyclin E1的蛋白表达量明显降低。结论CLSPN在胶质瘤中高表达,高水平CLSPN预示胶质瘤患者生存期较差。CLSPN通过调控了P53-p21/p27分子信号通路调控了细胞G0/G1期周期从而影响胶质母细胞瘤的增殖能力。CLSPN可能是一个潜在的胶质母细胞瘤治疗靶点。

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