NOB1影响胶质瘤细胞增殖、凋亡的实验研究

Effect of NOB1 on the proliferation and apoptosis of human glioma cells

目的利用RNA干扰(RNAi)在人胶质瘤U251和U87-MG细胞中沉默NOB1基因的表达,探讨NOB1对恶性胶质瘤细胞增殖以及凋亡的影响。方法构建NOB1基因的短发卡(shRNA)慢病毒表达载体,包装成病毒颗粒并感染人胶质瘤U251和U87-MG细胞,采用实时定量聚合酶链反应检测NOB1在恶性胶质瘤细胞系中的表达。采用甲基噻唑基四唑(MTT)比色法和克隆形成实验检测细胞增殖和克隆形成能力情况;同时利用PI染色流式细胞仪检测细胞凋亡情况,观察NOB1基因对U251和U87-MG细胞增殖、凋亡的影响。结果 NOB1基因在人胶质瘤U251、U87-MG细胞系中均明显高表达。NOB1-shRNA慢病毒能有效感染胶质瘤细胞,实验结果显示感染后U251和U87-MG细胞的增殖能力明显下降;U251克隆形成能力明显受到抑制;细胞周期在G0/G1停滞,而且细胞出现显著性凋亡;上述差异均具有统计学意义(P<0.05)。结论 NOB1在人胶质瘤U251和U87-MG细胞中高表达;降低NOB1基因的表达,可以显著降低胶质瘤细胞的增殖,并促进胶质瘤细胞凋亡;NOB1基因在体外对胶质瘤细胞的发生发展可能具有癌基因的调节作用。

Objective To explore the role of NOB1 （ NIN1/RPN12 binding protein 1 homolog）, as a ribosome assembly factor,effect the proliferation and apoptosis in human U251 and U87-MG glioma cells by RNA interfering. Methods Above all, construct the lentiviral vectors with NOB1 shRNA. Human glioma cell line U251 and U87-MG were infected by the lentiviral vectors encoding interfering RNA（RNAi） targeting the NOB1 gene. Real- time polymerase chain reaction （PCR） was performed to investigate the expression of NOB1 at mRNA levels. To further confirm the inhibitory effects of RNAi vectors on NOB1 gene expression, quantitative real-time RT-PCR and Western blotting assays were used. The proliferation of glioma cells after transduction with the NOB1-RNAi-Lentivirus （Lv-shNOB1）was evaluated by methyl thiazolyl tetrazolium （MTr）assay, colony formation and cell-cycle assays. Results NOB1 gene were significantly high expressed in human glioma U251, U87-MG cell lines. NOBI-shRNA Lentiviral could effectively infect glioma cells, the experimental results showed that the proliferation ability of U251 and U87-MG cells decreased after infection; the colony forming ability of U251 glioma cells was significantly inhibited;cells stagnated in the GO/G1 state of the cell cycle and cells apoptosised significantly; these differences were statistically significant（ P 〈 0. 05 ）. Conclusions NOB1 gene was highly expressed in human glioma U251 and U87-MG cells;the less expression of NOB1 could significantly reduced the proliferation and promote apoptosis of glioma cells ;In vitro ,NOB1 may regulate the occurrence and development of the glioma cells as an oncogene.