ABSTRACT
Objective To investigate the effect and mechanism of HPV16-E7 gene specific CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR associated system) on the cell apoptosis and proliferation of human cervical cancer SiHa cell line.Methods HPV16 positive cervical cancer SiHa cells and HPV16 negative human embryonic kidney HEK293 cells were cultured and each of the cells were divided into 3 groups respectively in the experiment:the control group was untreated,the C9 group was transfected only with Cas9 plasmid,and the Cri group was cotransfected with guide RNA (gRNA) plasmid and Cas9 plasmid (1∶3).T-7endonuclease Ⅰ assay was used to detect double strand break (DSB) formation in SiHa cells.The apoptosis rates of SiHa and HEK293 cells were detected by flow cytometry (FCM).CCK-8 was used to evaluate the proliferation of SiHa and HEK293 cells.Western blotting was applied to detect E7 and pRb protein expression.Results The SiHa cells in Cri group showed DSB formation at 48 h after transfection.The apoptosis rate of Cri group at 48 h was 26.6％,higher than 2.6％ in control group (x2 =5.455,P =0.020) and 3.1％ in C9 group (x2 =6.279,P =0.012).The apoptosis rates of control,C9 and Cri group were 7.4％,7.6％,7.9％ for HEK293 cells respectively.Compared with the control and C9 group,the apoptosis rate of the Crigroup showed no statistical significance (x2 =0.032,P =0.858;x2 =0.034,P =0.853).After 72 h transfection,the inhibition rate of SiHa cells in Cri group was 29.4％,higher than 15.0％ in control group (x2 =22.481,P =0.000) and 18.0％ in C9 group (x2 =24.879,P =0.000).The inhibition rate of the HEK293 cells in Cri group was 3.2％,and it showed no significant difference compared with the inhibition rate of C9 group (2.2％,x2 =2.857,P =0.091) and control group (2.3％,x2 =3.438,P =0.064) respectively.Down-regulated expression of E7 protein and up-regulated expression of pRb protein were detected of the SiHa cells in Cri group compared with the control group,while the E7 protein and pRb protein level did not show difference in the C9 group.Conclusion CRISPR specifically targeting HPV16-E7 oncogene can promote apoptosis of SiHa cells,and inhibit the cell proliferation.It is speculated that CRISPR system may induce DSB event of E7 gene,and result into increased expression of tumor suppressor protein pRb.