ABSTRACT
A trans-splicing ribozyme which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful agent for tumor-targeted gene therapy. In this study, we evaluated in vivo function of the hTERT-targeting trans-splicing ribozymes by employing the molecular analysis of expression level of genes affected by the ribozyme delivery into peritoneal carcinomatosis mice model. To this effect, we constructed adenoviral vector encoding the specific ribozyme. Noticeably, more than four-fold reduction in the level of hTERT RNA was observed in tumor nodules by the systemic infection of the ribozyme-encoding virus. Such hTERT RNA knockdown in vivo induced changes in the global gene expression profile, including the suppression of specific genes associated with anti-apoptosis including bcl2, and genes for angiogenesis and metastasis. In addition, specific trans-splicing reaction with the targeted hTERT RNA took place in the tumors established as peritoneal carcinomatosis in mice by systemic delivery of the ribozyme. In conclusion, this study demonstrates that an hTERT-specific RNA replacement approach using trans-splicing ribozyme represents a potential modality to treat cancer.
Subject(s)
Animals , Humans , Mice , Cell Line , Gene Expression/physiology , Genetic Vectors , Neoplasm Metastasis , Neoplasms/genetics , RNA, Catalytic/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Telomerase/antagonists & inhibitors , Trans-Splicing/geneticsABSTRACT
AIM: To determine the cleavage activity of anti-transforming growth factor ?1 hammerhead ribozymes which was inserted into U1 small nuclear RNA in cell-free system.METHODS: The hammerhead ribozyme targeting against transforming growth factor ?1 was designed through the analysis of computer software.The ribozyme fragments were synthesized and cloned into the U1 snRNA ribozyme vector pZeoU1EcoSpe,which contained U1 snRNA promoter/enhancer and terminator.TGF ?1 cDNA partial fragment was generated by RT-PCR,and then cloned into the T-vector at the downstream of T7 promoter.The transcripts of ribozyme and target RNA incorporated into isotope were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis.-labeled U1 snRNA chimeric ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: U1snRNA chimeric ribozyme(U1Rz803) cleaved TGF?1 mRNA efficiently and specifically at 37 ℃,while the disable ribozyme(U1Rz803m) showed no cleavage activity,so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possesses the perfect specific catalytic cleavage activity in cell-free system.These results indicate that U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGF?1 in vivo,therefore it may provide a new means for exploring the role of TGF?1 in hematopoietic regulation in the future.
ABSTRACT
AIM: To study the interrelation between the structure and the function of artificial ribozyme M_1GS. METHODS: Ribozyme M_1GS-T_7, which targeted the mRNA segment of HCMV UL54 gene, was constructed. The secondary structure of M_1GS was simulated under different temperatures (20 ℃, 37 ℃ or 55 ℃, at which the secondary structure of M_1GS was relatively stable) and the interrelation between the secondary structure and the cleavage activity of M_1GS was analyzed under different temperature in vitro. To investigate the interrelation between the structure and the function of ribozyme M_1GS further, mM_1GS-T_7 was designed, in which some mutation sites was added, according to the result of temperature change experiment and the simulated secondary structure, showing that were the same structures at 37 ℃ as that of M_1GS-T_7 at 55 ℃. RESULTS: In temperature change experiment, the cleavage activity of M_1GS-T_7 was highest at 55 ℃. The result of mutant experiment showed that the mutant type was more active than M_1GS-T_7 at 37 ℃. CONCLUSION: The cleavage activity of M_1GS, which has some certain secondary structure, is higher than others. There is some interrelation between the structure and the function of M_1GS.
ABSTRACT
AIM: To find out effective hammerhead ribozyme from the designed those in order to inhibit endothelin-1(ET-1) mRNA expression in ECV304 cells. METHODS: The sequences of hammerhead ribozyme were determined by computer. The eukaryotic expression vector pEGFP-R including ribozyme DNA was constructed and delivered into ECV304 cells by liposomes. The expression of ET-1 mRNA was examined by RT-PCR. RESULTS: Ribozyme 145 decreased the expression of ET-1 mRNA in ECV304 cells. CONCLUSION: ET-1 mRNA in ECV304 cells was degraded by R145.