Blocking activity of the HPV18 virus in cervical cancer cells using the CRISPR/Cas9 system

Objective: Specific sgRNA-sequences targeting oncogenes E6 and E7 in HPV18 were designed using the CRISPR/Cas9 system. These sgRNAs knocked out E6 and E7 expressions and were used to study their effects on the proliferation and cell cycle of the cervical cancer HeLa cell line.

Methods: Lentivirus vectors targeting E6 and E7 oncogenes were constructed and transfected into HeLa cells. mRNA and protein expression levels of E6 and E7 were measured by RT-PCR and Western blot, respectively. The cell cycle was detected by flow cytometry. A colony formation assay was applied to evaluate the proliferation capacity of the HeLa cells.

Results: Three E6 Cas9-sgRNA vectors targeting E6 and three E7 Cas9-sgRNA vectors targeting E7 genes were constructed and transfected into HeLa cells, respectively. RT-PCR results showed that all three E6 and E7 sgRNAs inhibited the expressions of E6 or E7 mRNA, respectively, when compared with the control groups. The inhibition ratios of the three groups of E6-sgRNAs were 28%, 85%, and 19%; the E7-sgRNAs were 86%, 25%, and 27%, respectively (P<0.05), with E6-sgRNA2 and E7-sgRNA1 having the greatest inhibitory effects. Western blot results showed that, compared with the control group, the protein expressions of E6 and E7 in the sgRNAs transfected group were also decreased, and E6-sgRNA2 and E7-sgRNA1 had the most inhibitory effects on E6 and E7 proteins. Flow cytometry results showed that the number of cells in G1/G0 was increased by 14.2% in the E6-sgRNA2 transfection group, and by 7.1% in the E7-sgRNA1 transfection group. Colony formation assay results showed that after transfection of E6 or E7 sgRNA plasmids, the HeLa cell colony was reduced significantly compared with the control group.

Conclusions: The CRISPR/Cas9 system targeting HPV18 E6 or E7 genes effectively blocked the transcription and expression of oncogenes E6 or E7 in HeLa cells, which resulted in cell cycle arrest and reduced cell proliferation.