应用CRISPR/Cas9靶向细胞内整合的HPV、基因治疗宫颈癌  

Inactivation of human papillomavirus E6 or E7 gene in cervical carcinoma cells by using CRISPR/Cas9 RNA-guided endonuclease

作　　者：陈思佳[1] 穆宇松 张林月[1] 钟照华[1] Chen Sijia;Mu Yusong;Zhang Linyue;Zhong Zhaohua(Department of Microbiology and Wu Lien-Teh Institute,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学微生物学教研室及伍连德研究所,150081

出　　处：《国际遗传学杂志》2020年第2期69-75,共7页International Journal of Genetics

基　　金：黑龙江省普通本科高等学校青年创新人才培养计划(UNPYSCT2015029)。

摘　　要：目的通过突变整合于宿主细胞中的人乳头瘤病毒(human papillomavirus,HPV)16型的E6、E7致癌基因,探索预防和治疗宫颈癌的新方法。方法以SiHa细胞系为研究对象,设计靶向其内整合的HPV16E6和E7基因的小引导RNA(small guide RNA,sgRNA),将其克隆入CRISPR/Cas9质粒,并应用其将E6、E7基因敲除,随后应用错位酶切法和克隆测序检测突变效果,并用细胞迁移与侵袭实验检测细胞的侵袭力。结果设计的sgRNA被克隆入CRISPR/Cas9质粒,经测序克隆正确。该质粒转染SiHa细胞后,可以引发靶点DNA的突变,致使E6和E7基因密码子改变,无法正确表达。E6、E7突变后SiHa细胞侵袭及增殖能力下降(t检验,pCas9组与psgE6-1-1-Cas9组相比,P=0.0006;pCas9组与psgE7-1-2-Cas9组相比,P=0.0007),促进肿瘤抑制因子P53的表达恢复,细胞的恶性度降低。Objective To explore the new method of preventing and treating cervical cancer by mutating oncogenes E6 and E7 of human papillomavirus(HPV)16 integrated into the host cells.Methods SiHa cell line was used as the research object.E6 and E7 genes were mutated by small Guided RNA(sgRNA)targeting HPV16 E6 and E7 genes using CRISPR/Cas9 technology.The effect of mutation was detected by dislocation enzyme digestion and clone sequencing.The invasiveness of cells was detected by the transwell test.Results The designed gRNA was successfully cloned into a CRISPR plasmid and detected by gene sequencing.The co-transfection of the plasmid and the plasmid expressing Cas9 into SiHa cells can induce the changes in the target DNA,leading to changes in the codons of E6 and E7 genes.Therefore,the E6 and E7 genes cannot be correctly expressed,and the expression of tumor suppressor P53 can be restored.The invasiveness and malignancy of SiHa cells were reduced after mutating E6 and E7 genes(t test,pCas9 group vs psgE6-1-1-Cas9 group,P=0.0006;pCas9group vs psgE7-1-2-Cas9 group,P=0.0007).

关 键 词：CRISPR/Cas9 HPVE6 HPVE7 宫颈癌治疗 

分 类 号：R737[医药卫生—肿瘤]