宫颈癌细胞PAX1基因甲基化的CRISPR靶向调控系统的构建

Construction of CRISPR-mediated targeted regulation system for PAX1 methylation in cervical cancer cell

目的:建立PAX1基因甲基化的CRISPR靶向调控系统。方法:首先,通过甲基化特异性PCR(MSP)测定宫颈癌细胞系(CaSki,SiHa)及正常细胞(人胚肾细胞HEK293T)中PAX1基因启动子的DNA甲基化水平;通过荧光定量PCR(RT-PCR)和免疫印迹(Western blotting)分别检测上述细胞系的mRNA及蛋白表达。然后,在PAX1高甲基化的宫颈癌细胞共转染sgRNA(single guide RNA)和dCas9-Tet1质粒,采用焦磷酸测序(Pyrosequencing)和RT-PCR分别检测其基因甲基化和表达水平的改变。最后,结合生物信息学预测和RT-PCR评估该CRISPR系统的脱靶效应。结果:与HEK293T细胞相比,宫颈癌细胞的PAX1基因启动子呈现高甲基化,且其mRNA(CaSki:t=9.13;SiHa:t=12.31;P<0.05)和蛋白表达水平(CaSki:t=16.72;SiHa:t=11.81;P<0.05)均降低。在CaSki及SiHa细胞系中,去甲基化sgRNA3(act-sgRNA3)组调控最显著,其PAX1基因的甲基化水平在CaSki和SiHa细胞系中分别下降了22.21%(t=6.65,P<0.05)、19.62%(t=17.00,P<0.05),其mRNA表达水平均显著上调(P<0.05)。且该系统脱靶效应基因的表达差异均小于2倍。结论:本研究成功建立了PAX1基因甲基化的CRISPR靶向调控系统,可有效的降低其甲基化水平,增强其内源性的表达水平。

Objective:To establish a CRISPR-mediated targeted regulation system for PAX1 methylation.Methods:Firstly,methylation-specific PCR(MSP)was performed to examinate the DNA methylation level of PAX1 promoter in cervical cancer cells(CaSki,SiHa)and normal cells(human embryonic kidney cell,HEK293T).The mRNA and protein expression of these cell lines was detected by real time quantitative PCR(RT-PCR)and Western blotting.Secondly,single guide RNA(sgRNA)and dCas9-Tet1 plasmid were co-transfected into PAX1 hypermethylated cells,and the changes in methylation and expression were detected via Pyrosequencing and RT-PCR,respectively.Finally,the off-target effect of the CRISPR system was evaluated by bioinformatics prediction and RT-PCR.Results:Compared with HEK293T cells,the methylation level of PAX1 promoter was hypermethylated in cervical cancer cells,and the expression levels of mRNA(CaSki:t=9.13,SiHa:t=12.31,P<0.05)and protein(CaSki:t=16.72,SiHa:t=11.81,P<0.05)was decreased.In CaSki and SiHa cell lines,demethylated sgRNA3(act sgRNA3)group had the most significant regulation.The methylation level of PAX1 promoter in the sgRNA3 group(act-sgRNA3)was decreased by 22.21%(t=6.65,P<0.05)and 19.62%(t=17.00,P<0.05)in CaSki and SiHa cell lines,respectively,and the mRNA expression levels were significantly up-regulates(P<0.05).The difference of off-target effect on gene expression was less than 2-fold.Conclusion:Our study successfully establishes CRISPR-mediated targeted regulation system for PAX1 methylation,which effectively downregulats the methylation level by increasing the endogenous expression level.