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Stable expression of anti-HPV 16 E7-ribozyme in CV-1 cell lines
1Department of Biochemistry, First Military Medical University, Guangzhou, China.
Abstract:
The HPV16 (human papilloma virus type 16) E7 gene product, an oncoprotein, has been considered to be involved in the pathogenesis of anogenital cancer, particularly of cervical cancer. In order to evaluate the effect of suppression of the expression of the E7 gene in CV-1 cells by ribozyme, Rz523 with a transacting ribozyme targeted to the E7 RNA and two processing ribozyme genes at the 5' and 3' flank was cloned into the eukaryotic expression plasmid pREP9 under the control of RSV-LTR promoter. The resultant plasmid pRSV-Rz523 was transfected into CV-1 cells by calcium phosphate coprecipitation. The expression of the ribozyme in G418-resistant cells was detected by dot-blot hybridization. Ribozymes stably expressed in the CV-1 cells were at a level of 9.0 pmol per 10(6) cells, in which the active ribozyme molecules were more than 50 fmol per 10(6) cells. The result of RNase protection assay showed that the steady-state level of the E7 RNA fragment in CV-1 cell lines was significantly reduced by about 90% in ribozyme-expressing cells. In contrast, the antisense control plasmid pRSV-AE7 only exhibited about 20%. This result implicated the possibility of reversing the malignant phenotype of cervical cancer by means of suppressing the expression of the E7 gene with ribozyme.
Insights
This study demonstrates that ribozymes can effectively suppress the human papilloma virus type 16 (HPV16) E7 gene, a key factor in cervical cancer development. This suppression offers a potential therapeutic strategy for reversing cancer progression.
Area of Science:
- Molecular Biology
- Oncology
- Virology
Background:
- The human papilloma virus type 16 (HPV16) E7 oncoprotein is implicated in anogenital cancers, particularly cervical cancer.
- Targeting the E7 gene offers a potential strategy for cancer therapy.
Purpose of the Study:
- To evaluate the efficacy of a ribozyme (Rz523) in suppressing HPV16 E7 gene expression in CV-1 cells.
- To assess the potential of ribozyme-mediated gene suppression for reversing malignant phenotypes.
Main Methods:
- Cloning of Rz523 into the pREP9 plasmid under the RSV-LTR promoter.
- Transfection of CV-1 cells using calcium phosphate coprecipitation.
- Detection of ribozyme expression via dot-blot hybridization and RNase protection assay.
Main Results:
- Stable expression of Rz523 in CV-1 cells reached 9.0 pmol per 10(6) cells, with active molecules exceeding 50 fmol per 10(6) cells.
- Ribozyme expression significantly reduced E7 RNA levels by approximately 90%.
- An antisense control plasmid showed only a 20% reduction in E7 RNA levels.
Conclusions:
- Ribozyme Rz523 effectively suppresses HPV16 E7 gene expression in vitro.
- This ribozyme-mediated gene silencing demonstrates potential for therapeutic intervention in cervical cancer.