[Antisense targeting to human papillomavirus 18 E6/E7 affects the proliferation and apoptosis of human cervical carcinoma: an in vitro experiment with HeLa cells]

Abstract

Objective: To investigate the effect of eukaryotic fluorescent expression vector carrying antisense human papillomavirus (HPV) 18 E6/E7 on the growth and proliferation of human cervical carcinoma.

Methods: The HPV18 E6E7 with the length of 716 bp was amplified by PCR, the PCR product was inversely inserted into the eukaryotic fluorescent expression vector pEGFP-C1 so as to construct the recombinant eukaryotic expression plasmid pEGFP-HPV18E6E7as (EGFP-18AS). Human cervical carcinoma cells of the line HeLa were cultured and randomly divided into 3 groups: Group A transfected with the recombinant plasmid EGFP-18AS, Group B transfected with the blank plasmid pEGFP-C1, and Group C without transfection used as control group. The mRNA expression of HPV 18 E6/E7 in the HeLa cells was detected by RT-PCR and protein expression of HPV18 E6/E7 HPV 18 E6/E7 in the HeLa cells was detected by. Western blotting MTT assay was performed to dynamically monitor the surviving cells and the cell apoptosis was observed by flow cytometry and fluorescence microscopy.

Results: The protein and mRNA expression levels of HPV18 E6/E7 in the HeLa cells transfected with HeLa/18AS were both remarkably lower than those in the HeLa cells transfected with blank plasmid and those of the control group. The numbers of surviving HeLa cells of Group A was significantly lower than those of Groups B and C (both P < 0.05). The phenomenon of arrest of G(1) phase was remarkable in Group A. The apoptotic rate of the cells of Group A was 47.21%, significantly higher than those of Groups B and C (14.18% and 3.36% respectively, both P < 0.05). An increased number of cells with chromosome condensation and fragmentation was found in Group A as compared with Groups B and C.

Conclusion: The recombinant pEGFP-HPV18E6E7as can effectively inhibit the growth and proliferation of human cervical carcinoma HeLa cells, and further induce the cell apoptosis. The antisense RNA technology is available and may provide a new way to gene therapy of the cervical carcinoma.