Transfection with the ribozyme targeting HPVE6 mRNA results in growth inhibition of E6-expressing cervical carcinoma cells

摘要

Objective: To acquire a ribozyme against the E6 gene of human papillomaviruses type 16 (HPV16E6) and investigate its effects on the phenotypes and gene expression of cervical cancer cell line. Methods: Anti-HPV16E6 ribozyme (HRz) was designed by computer programs and its activity identified by cleavage experiment in vitro before its transfection via lipofectin into CaSKi cells with the empty eucaryotic expression plasmid transfection of the cells also performed, the resultant cells designated as CaSKi-R, CaSKi-P respectively. The morphology and the soft agar forming ability were studied in CaSKi cells and the transfected cells, and the expression of E6, proliferating cell nuclear antigen (PCNA) and C-erbB-2 genes assayed by flow cytometry. The tumorgenicity of each cell line was evaluated in nude mice receiving inoculations of CaSKi, CaSKi-R and CaSKi-P cells separately, while in one group, both CaSKi and CaSKi-R cells were inoculated on different sides of the mice. Results: HRz was able to cleave HPV16E6 mRNA in a site-specific manner and could be expressed stably in transfected CaSKi cells. Northern blot analysis showed that E6 mRNA was less in CaSKi-R than in CaSKi cells, and no significant difference in the morphology and growth rate was observed between CaSKi and CaSKi-P cells, but the growth rate CaSKi-R was lowered. The colony-forming rate of CaSKi-P in soft agar was similar to that of CaSKi cells, while that of CaSKi-R was decreased. Flow cytometry showed that anti-HPV16E6 ribozyme reduced the expression of E6, PCNA and C-erbB-2 genes in CaSKi-R cells, but not in CaSKi-P cells. The tumorgenicity of CaSKi-R in nude mice was decreased compared with CaSKi cells. Conclusion: HRz can partially reverse the malignant phenotype of CaSKi cells, possibly due to decreased E6 gene expression, and the consequent decrease of PCNA and C-erbB-2 gene expressions.