Syndecan-1基因敲低抑制胶质瘤U87细胞的增殖

Syndecan-1 knockdown inhibits the proliferation of U87 glioblastoma multiforme cell

目的探讨多配体蛋白多糖-1(SDC1)在不同级别胶质瘤标本中的表达水平及其敲低对U87细胞的增殖的影响。方法通过提取癌症和肿瘤基因图谱(TCGA)和美国国立生物信息中心基因表达文库(NCBI-GEO)两大数据库中胶质瘤标本的信息,分析Syndecan-1的表达与胶质瘤的等级和胶质瘤患者的预后的关系;将携带SDC1 shRNA的慢病毒载体感染U87细胞,稳定敲低的细胞株为干扰组,未转染为空白对照组,转染scramble序列为阴性对照组;采用MTT法、平板克隆、台盼蓝拒然法和流式细胞术检测细胞增殖能力;qRT-PCR和Western blot检测相关蛋白变化。结果SDC1的表达与胶质瘤的等级成正相关(P<0.0001),与胶质瘤患者的预后负相关(P=0)。成功构建稳定敲低SDC1的U87细胞株;与空白和阴性对照组相比,MTT实验显示干扰组细胞细胞活性明显降低(P<0.01),平板克隆能力明显受到抑制(P<0.01),生长曲线提示增殖能力降低(P<0.05),流式细胞实验提示干扰组细胞周期被抑制在G0/G1期,S期细胞数明显减少,其增殖指数((S+G2)/(G1+S+G2))分别为(P<0.01);SDC1和PCNA的mRNA和蛋白表达水平明显降低(P<0.05)。结论SDC1的表达随着胶质瘤的等级增加而表达增强,其高表达提示胶质瘤患者不良预后;SDC1基因的敲低可抑制胶质瘤U87细胞的增殖,提示SDC1可能成为胶质瘤生物治疗的新靶点。

Objective To determine the expression of syndecan-1(SDC1)in glioma and its function on U87 glioma cell.Methods The expression of syndecan-1 in glioma was analyzed in the data from The Cancer Genome Atlas(TCGA)and The National Center for Biotechnology Information Gene Expression Omnibus(NCBI-GEO);U87 cells were transfected with lentiviral vector carrying SDC1 shRNA to construct a stable SDC1-silencing cell line;the cell proliferation was analyzed by MTT assay,trypan blue exclusion assay and the colony formation assays repectively.The flow cytometry was used to determine the proliferation indices in shSDC1 group;the expression of SDC1 and Proliferation Cell Nuclear Antigen(PCNA)at mRNA and protein levels were detected by qRT-PCR and Western blotting.Results Both TCGA and NCBI-GEO show that high transcription level of SDC1,on average,is correlated with advanced tumor grade(Kruskal-Wallis non-parametric test,P<0.0001 in both datasets).Moreover,elevated SDC1 expression indicates clinical significance,as shown by analysis of TCGA patient survival segregated by SDC1 expression.Glioma patients with SDC1 expression level above two-third showed a great decrease in survival with regard to those with SDC1 expression level below one-third(Kaplan-Meier survival analysis,P=0 in both log-rank and Wilcoxon statistical tests).The stable SDC1-silencing cell line was successfully established;Compared with control and scramble groups,MTT assay showed that the viability of shSDC1 group in U87 cells was significantly suppressed after 72h culture(0.56±0.04vs0.74±0.02、0.74±0.02,P<0.01),and the growth curve was significantly delayed in a time-dependent manner in shSDC1 group(P<0.05).In addition,the colony formation assays showed a significant inhibition of cell proliferation and colony formation(39.33±9.61vs72.67±1.53、69.00±6.00,P<0.01).The proliferation indices[(S+G2)/(G1+S+G2)]of shSDC1 group decreased significantly in comparison with the control group and scramble group(35.64%±5.24%,47.20%±0.92%,45.69%±1.44%,P<0.01).At the same time,the expression of SDC1 and PCNA at mRNA and protein levels was reduced significantly(P<0.05).Conclusion The elevated syndecan-1expression in glioma is closely associated with advanced tumor progression and shortened survival,implying that SDC1 might serve as a novel target in the biotherapy of glioma.