Stable expression of anti-HPV 16 E7-ribozyme in CV-1 cell lines.
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.
Huang
Y
Y
Department of Biochemistry, First Military Medical University, Guangzhou, China.
Kong
Y
Y
Wang
Y
Y
Qi
G
G
Lu
C
C
D002460:Cell Line; D003001:Cloning, Molecular; D006868:Hydrolysis; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D010957:Plasmids; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic; D012367:RNA, Viral; C059731:oncogene protein E7, Human papillomavirus type 16