[Hammerhead ribozyme against human telomerase catalytic subunit (hTERT) induced apoptosis of liver cancer cells].
OBJECTIVE
To construct vector pEGFP-C1-hTERT-ribozyme (pGTRz-U6) and its mutant (pGTmRz-U6) against hTERT containing U6 promoter, then transfect them into human liver cancer cell line SMMC7721 to observe the action of the human telomerase catalytic subunit (hTERT) hammerhead ribozyme on proliferation and apoptosis of human liver cancer cell SMMC7721.


METHODS
Eukaryotic expressing vector pGTRz-U6 and mutant pGTmRz-U6 were constructed and transfected into SMMC7721 using Lipofectamine2000 Reagent, with pEGFP-C1 as the control group. After strict screening by G418, positive clones were cultured; the amount of expression of ribozyme and hTERT was detected by RT-PCR; cell proliferation by MTT; telomerase activity by TRAP and silver staining assay; cell apoptosis by FCM.


RESULTS
We found that the two ribozymes were expressed persistently in SMMC7721; different expression levels (P < 0.01) of hTERT among SMMC7721-Rz, SMMC7721-mRz and SMMC7721-pEGFP-C1 was exhibited by the analysis of variance with SPSS software. The difference between SMMC7721-Rz and the others is significant in t-test (P < 0.01), while there was no difference between SMMC7721-mRz and SMMC7721-pEGFP-C1 (P > 0.05). With the advance of cell division, telomerase activities of the cells treated by SMMC7721-Rz and SMMC7721-mRz decreased gradually, and the percentage of apoptosis of the cells transfected with Rz and mRz increased gradually. The apoptosis percentage of 7PDS SMMC7721-Rz was 29.86%, while those of SMMC7721-mRz and SMMC7721-pEGFP-C1 were 9.87% and 3.36%, respectively.


CONCLUSIONS
The apoptosis level of SMMC7721 induced by hTERT ribozyme increases as cells divide, and this ribozyme maybe a potential approach for liver cancer gene therapy.
Song
Dong-po
DP
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Lin
Ju-sheng
JS
Fu
Gui-lian
GL
Sun
Xue-mei
XM
Kong
Xin-juan
XJ
Li
Pei-yuan
PY
Ma
Xin
X
D017209:Apoptosis / Q000502:physiology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D009154:Mutation; D016337:RNA, Catalytic / Q000235:genetics / Q000494:pharmacology*; D019098:Telomerase / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection
D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D019098:Telomerase