抗端粒酶核酶质粒的构建筛选及其对CNE-2Z细胞增殖与凋亡的影响

Construction and Screening of a Ribozyme Targeting Telomerase RNA and Effects of Telomerase Ribozyme on Proliferation and Apoptosis of CNE-2Z Cells

背景与目的:已证实端粒酶(telomerase,TLMA)对肿瘤的进展和肿瘤细胞的无限增殖起着重要的决定作用。核酶是具有特殊核酸内切酶活性的反义RNA,可序列特异性地与靶RNA分子配对并切割靶基因RNA。有报道人低分化鼻咽癌CNE-2Z细胞端粒酶阳性,本实验目的是构建抗端粒酶RNA模板区特异性核酶的真核表达载体并用电穿孔法将其导入人低分化鼻咽癌CNE-2Z细胞,研究该核酶对CNE-2Z细胞增殖、凋亡的影响。方法:设计合成针对端粒酶RNA模板区的锤头状核酶基因teloRZ作为端粒酶抑制剂,构建3种带有绿色荧光蛋白(greenfluorescentprotein,GFP)报道基因和嘌呤霉素(puromycin)抗性基因的teloRZ真核表达质粒pGFPuro-teloRZ2.1、pGFPuro-teloRZ7.1、pGFPuro-teloRZ7.7,这3种质粒的不同点在于teloRZ基因和puromycin抗性基因按3种不同方向设计,然后将上述3种质粒及载体质粒pPAT-GFP电转染CNE-2Z细胞,用荧光显微镜检测GFP表达情况;用流式细胞仪、荧光染色法检测细胞增殖指数及凋亡等指标。结果:CNE-2ZGTR7.1细胞(转染目的基因质粒pGFPuro-teloRZ7.1的CNE-2Z细胞)增殖指数(25.100±0.141)%明显低于CNE-2Z细胞(未转染质粒的细胞)的(53.663±16.981)%、CNE-2ZG细胞(转染空载质粒pPAT-GFP的CNE-2Z细胞)的(61.575±5.166)%、CNE-2ZGTR2.

BACKGROUND &OBJECTIVE: It was proved that telomerase is an importa nt determinant in tumor progression and cell immortalization. Ribozyme is a spec ial kind of trans-acting RNA with endonuclease activity and sequence-specific catalytic RNA molecules, which can cleave target RNA. It was reported that telom erase activity is present in human poorly-differentiated nasopharyngeal carcino ma (NPC) CNE-2Z cells. This study was designed to construct eukaryotic expressi on plasmids containing telomerase ribozyme (teloRZ)gene targeting the template region of human telomerase RNA (hTR) and then to transfect the plasmids into CN E-2Z cells by electroporation to investigate the effect of teloRZ on proliferat ion and apoptosis of those transfected CNE-2Z cells. METHODS: Hammer ribozyme g ene teloRZ directed against telomerase RNA templet was designed and synthesized to serve as a telomerase inhibitor. Three different eukaryotic expression plasmi ds carried with the green fluorescent protein (GFP) reporter gene and puromycin -resistance gene and containing teloRZ gene were constructed. They were referre d to as pGFPuro-teloRZ2.1, pGFPuro-teloRZ7.1, and pGFPuro-teloRZ7.7 and diffe red in the relative orientation of the genes for telomerase-ribozyme and puromy cin-resistance. The CNE-2Z cells were transfected with three expression plasmi ds and control plasmid pPAT-GFP by electroporation. The expression of GFP was d etected by fluorescent microscope; cellular proliferation index (PI) and apoptos is were investigated by flow cytometry analysis and fluorescence staining. RESUL TS: PI of CNE-2ZGTR7.1 cells transfected by plasmid pGFPuro-teloRZ7.1 (25.100 %±0.141%)was significantly lower than those of CNE-2Z cells untransfected b y any plasmid (53.663%±16.981%),CNE-2ZG cells transfected by control plasmid pPAT-GFP (61.575%±5.166%),CNE-2ZGTR2.1 cells transfected by plasmid pGFPur o-teloRZ2.1 (61.500%±20.082%), and CNE-2ZGTR7.7 cells transfected by plasmi d pGFPuro-teloRZ7.7 (59.400%±13.933%) (P< 0.01). GFP was detected in CNE-2Z G cells,CNE-2ZGTR7.1 cells, and CNE-2ZGTR7.7 cells;while there was no GFP expr ession in CNE-2Z cells and CNE-2ZGTR2.1 cells. The plasmid pGFPuro-teloRZ7.1 was selected from 3 plasmids for further experiments. Apoptosis could be observe d in CNE-2ZGTR7.1 cells after 12 generations. There was no apoptosis occurring in CNE-2Z and CNE-2ZG cells. CONCLUSION: The teloRZ7.1 gene was electroporated successfully into CNE-2Z cells. TeloRZ7.1 can inhibit the proliferation and in duce apoptosis of CNE-2Z cells. These findings suggest the potential applicatio n of ribozyme teloRZ7.1 as telomerase inhibitor.