The Effects Of Anti-Hpv16 E6-Ribozyme On The Cellproliferation And Invasiveness Of Cervical Carcinoma-CaSKi Cell Line

Ribozymes are unique RNA molecules with enzymatic activity which is able to cleave targeted RNA substrates site-specifically. It has been identified that ribozymes are effective modulators of gene expression, such as activated oncogene or foreign virus gene. In comparison with antisense RNA, ribozymes can not only block but also cleave target RNAs. Moreover, they cleave the RNAs in a recycling manner, which means the ribozyme can dissociate from the ribo-RNA complex and bind to a new RNA molecule after cleaving one target molecule. It has been proved that the secondary structure of ribozymes is stabler and simpler than single-strand antisense RNA, which makes them more insensitive to the nuclease. In addition, ribozymes can also be artificially designed. For all these reasons, the discovery of the ribozymes provide more specific methods of gene therapy on the human cancer. At present, ribozymes commonly used in gene therapy are hammerhead and hairpin types.Recent studies revealed that COX-2, VEGF and MMP-9 were overexpressed in cervical cancer, and their expression levels were closely related to the prognosis of the patients. So it may be necessary to block the overexpression of COX-2, VEGF and MMP-9 in tumor cells with ribozyme technique, thus to prevent the cells from over proliferation, and-5-search for a new treatment of cervical cancer. In this study, we performed the following experiments:1.By means of lipofectin transfection, Anti-HPV16 E6-ribozyme and empty eukaryotic expressing plasmids were transfected into CaSKicell line, which were named as CaSKi-R, CaSKi-P respectively. The expressions of anti-HPV16 E6-ribozyme and HPV16E6 in the three cervical cancer cells were detected and compared by Dot blot of anti-HPV16 E6-ribozyme probe and Northern blot of HPV16E6 probe, both tagged with 32P. Anti-HPV16 E6-ribozyme and HPV16E6 genes were proved to be steadily expressed in CaSKi-R cells by Northern blot and Dot blot.2. CaSKi, CaSKi-R and CaSKi-P were studied in their cell growth curves, soft agar forming ability, cell invasiveness and tumorgenicity innude mice. The results showed that there is no distinct difference of the growth rate, the soft agar forming ability, the cell invasiveness and tumorgenicity between CaSKi and CaSKi-P. Compared with them, the growth rate, the soft agar forming ability, the cell invasiveness and tumorgenicity of CaSKi-R evidently decreased.3. The expression of COX-2, VEGF and MMP-9 mRNA were detected by one-step RT-PCR . The result of RT-PCR analysis showed that the expression levels of COX-2, MMP-9 and VEGF mRNA on CaSKi-R cells were lower than those on CaSKi-P and CaSKi cells.4. The antigen expression of COX-2, VEGF and MMP-9 were detected by immunocytochemistry. The antigen expressions of COX-2, MMP-9 and VEGF were found in all three kinds of CaSKi cells by immunocytochemistry. But, the antigen expressions of COX-2, MMP-9 and VEGF in CaSKi-R were weaker than those in CaSKi and CaSKi-P cells.To sum up, Anti-HPV16 E6-ribozyme could to some extent reduce the ability of proliferation and invasiveness of cervical cancer cell line CaSKi, which may be related to the decrease of COX-2, MMP-9 and VEGF-6-expression in CaSKi cells.