Disruption of HPV16-E7 by CRISPR/Cas System Induces Apoptosis and Growth Inhibition in HPV16 Positive Human Cervical Cancer Cells
Figure 4
Effect of HPV16-E7 gRNA-4/Cas9 on cellular proliferation, DSB cleavage, and E7 and pRb proteins level. HPV16-E7 gRNA-4/Cas9 inhibited cellular viability in SiHa and Caski cells but not in C33A and HEK293 cells ((a)–(d)). Cellular viability of SiHa, Caski, C33A, and HEK293 cells were monitored by CCK-8 assay ( compared to untreated group, per Student’s -test). T7E1 treatment of heteroduplex DNA in the gRNA-4/Cas9 group showed noncleaved products at 500 bp and cleaved products at 300 bp and 200 bp both in SiHa and Caski cells ((e), (f)). Black arrows indicate 500 bp, 300 bp, and 200 bp PCR products. Untreated represented cells transfected with only Cas9 plasmid and was used as a negative control. Con-gRNA meant cells treated with HPV16E6-gRNA-1/Cas9, which was proved to be inactive preexperimentally. was 100 bp DNA marker. E7 protein was downregulated and pRb protein was upregulated both in SiHa and Caski cells ((g), (h)). Data were drawn from four independent experiments. β-Actin was used as an internal control.
