[Construction and screening of a ribozyme targeting telomerase RNA and effects of telomerase ribozyme on proliferation and apoptosis of CNE-2Z cells].
OBJECTIVE
It was proved that telomerase is an important determinant in tumor progression and cell immortalization. Ribozyme is a special kind of trans-acting RNA with endonuclease activity and sequence-specific catalytic RNA molecules, which can cleave target RNA. It was reported that telomerase activity is present in human poorly-differentiated nasopharyngeal carcinoma (NPC) CNE-2Z cells. This study was designed to construct eukaryotic expression plasmids containing telomerase ribozyme (teloRZ)gene targeting the template region of human telomerase RNA (hTR) and then to transfect the plasmids into CNE-2Z cells by electroporation to investigate the effect of teloRZ on proliferation and apoptosis of those transfected CNE-2Z cells.


METHODS
Hammer ribozyme gene teloRZ directed against telomerase RNA templet was designed and synthesized to serve as a telomerase inhibitor. Three different eukaryotic expression plasmids carried with the green fluorescent protein (GFP) reporter gene and puromycin-resistance gene and containing teloRZ gene were constructed. They were referred to as pGFPuro-teloRZ2.1, pGFPuro-teloRZ7.1, and pGFPuro-teloRZ7.7 and differed in the relative orientation of the genes for telomerase-ribozyme and puromycin-resistance. The CNE-2Z cells were transfected with three expression plasmids and control plasmid pPAT-GFP by electroporation. The expression of GFP was detected by fluorescent microscope; cellular proliferation index (PI) and apoptosis were investigated by flow cytometry analysis and fluorescence staining.


RESULTS
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 by 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 pGFPuro-teloRZ2.1 (61.500%+/-20.082%), and CNE-2ZGTR7.7 cells transfected by plasmid pGFPuro-teloRZ7.7 (59.400%+/-13.933%) (P< 0.01). GFP was detected in CNE-2ZG cells,CNE-2ZGTR7.1 cells, and CNE-2ZGTR7.7 cells;while there was no GFP expression 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 observed in CNE-2ZGTR7.1 cells after 12 generations. There was no apoptosis occurring in CNE-2Z and CNE-2ZG cells.


CONCLUSIONS
The teloRZ7.1 gene was electroporated successfully into CNE-2Z cells. TeloRZ7.1 can inhibit the proliferation and induce apoptosis of CNE-2Z cells. These findings suggest the potential application of ribozyme teloRZ7.1 as telomerase inhibitor.
Zhao
Ying-Hai
YH
Department of Pathology, Guangdong Medical College, Zhanjiang, Guangdong, 524023, P.R.China.
Chen
Xiao-Yi
XY
R Arrand
John
J
D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000473:pathology*; D010957:Plasmids; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000494:pharmacology*; D019098:Telomerase / Q000378:metabolism*; D014162:Transfection
D016337:RNA, Catalytic; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase