摘要
目的探讨小干扰RNA(siRNA)沉默泛素特异性肽酶22(ubiquitin specific protease 22,USP22)对人神经母细胞瘤细胞增殖的影响和作用机制。方法合成USP22 siRNA及阴性对照siRNA(control siRNA),用脂质体Lipofectamine 2000转染至人神经母细胞瘤SH-SY5Y细胞和SK-NSH细胞,且设立阴性对照组和实验组。通过Western blot法和逆转录聚合酶链反应(RT-PCR)检测两组神经母细胞瘤细胞的USP22的蛋白表达和mRNA表达水平的变化。采用四甲基偶氮唑蓝比色法(MTT法)检测USP22siRNA对两组神经母细胞瘤细胞的增殖抑制率。通过原位凋亡TUNEL法检测USP22 siRNA对两组神经母细胞瘤细胞凋亡情况。流式细胞仪定量检测USP22 siRNA对神经母细胞瘤细胞凋亡及细胞周期分布的影响。结果与设立的阴性对照组相比较,实验组中神经母细胞瘤细胞中USP22蛋白和mRNA表达显著下降(P <0. 05),神经母细胞瘤细胞增殖明显受到抑制(P <0. 05),凋亡率随时间延长明显上升(P <0. 05),细胞周期中G2/M期的细胞比例明显增高。结论 USP22基因在神经母细胞瘤细胞的增殖中发挥重要的作用,采用siRNA技术能够有效地沉默USP22基因的表达,并显著抑制人神经母细胞瘤细胞的增殖,其机制可能与调节细胞周期及细胞凋亡相关。
Objective To explore the influence of ubiquitin-specific protease 22 (USP22) gene silencing by small interference RNA (siRNA) on proliferation of human neuroblastoma cells and its mechanism. Methods USP22 siRNA and a negative control siRNA were designed and transfected into neuroblastoma cell lines SH-SYSY and SK-NSH by using Lipofectamine 2000. The cells which were transfected with USP22 siRNA and control siRNA were set as experimental and negative control groups respectively. RT-PCR and Western blot were used to detect the expressions of USP22 mRNA and protein in neuroblastoma cells in two groups. Cell proliferation was measured by MTT. The apoptosis of cells was determined by TUNEL. The apoptosis rate and the distribution of cell cycle of SH-SYSY and SK-NSH were determined by flow cytometry. Results Compare with negative control group, the expressions of USP mRNA and protein were decreased significantly ( P 〈 0. 05 ), cell proliferation was obviously inhibited (P 〈 0. 05 ), apoptosis rate rose markedly (P 〈 0. 05), the proportion of G2/M in cell cycle was higher (P 〈 0. 05). Conclusion USP22 gene plays a key role in the proliferation of human neuroblastoma cells by regulating the apoptosis and cell cycle.
作者
曲志博
鞠春成
刘家旭
马计超
周鑫
刘冰洋
张浩然
孙清政
张世伟
刘彦龙
QU Zhi-bo;JU Chun-cheng;LIU Jia-xu;MA Ji-chao;ZHOU Xin;LIU Bing-yang;ZHANG Hao-ran;SUN Qing-zheng;ZHANG Shi-wei;LIU Yan-long(Department of General Surgery,Harbin Children Hospital,Harbin 150010,China;Department of Colorectal Surgery,Cancer Hospital of Harbin Medical University,Harbin 150081,China)
出处
《哈尔滨医科大学学报》
CAS
2018年第3期216-220,225,共6页
Journal of Harbin Medical University
基金
哈尔滨市科技局科技创新人才研究专项基金后备人才项目(2014RFQGJ124)
关键词
USP22
SIRNA
神经母细胞瘤
细胞凋亡
ubiquitin specific protease 22
small interference RNA
neuroblastoma
cell apoptosis