CRISPRi靶向调控HCV复制相关miR-122的表达

Regulation of expression of HCV replication associated mi R-122 by CRISPR interference

目的:探索CRISPR干扰(CRISPR interference,CRISPRi)特异性抑制肝癌细胞Hep G2内源性miR-122表达.方法:设计靶向mi R-122启动子区TATA盒和转录起始位点(transcription start site,TSS)的sg RNA(sg T1和sg T2),与无DNA内切酶活性仅保留识别特性的d Cas9-KRAB载体共转染Hep G2细胞,通过实时定量PCR(quantitative Real-time PCR,qR T-PCR)方法检测miR-122的表达并确立有效的sgR NA;设计不同的sgR NA浓度,探索CRISPRi抑制作用的剂量依赖性;通过qR T-PCR及Western blot方法检测miR-122靶分子HOMX1和CyclinG 1的表达变化.结果:sg T1和sg T2介导的CRISPRi分别将肝癌细胞Hep G2内源性mi R-122的表达水平抑制5.5倍(P<0.01)和3.7倍(P<0.01);CRIPSRi抑制效应是sgR NA剂量依赖性的,当lentiG uidePuro-sgT 1质粒为500 ng时,可将miR-122的表达抑制10.0倍(P<0.01);CRIPSRi抑制肝癌细胞Hep G2内源性mi R-122表达的同时,上调了mi R-122下游靶分子HMOX1和Cyclin G1的表达.结论:本研究利用CRISPRi技术实现了对肝癌细胞Hep G2内源性mi R-122表达的特异性抑制,为抗丙型肝炎病毒(hepatitis C virus,HCV)提供了全新的策略.

AIM： To investigate whether clustered regularly interspaced short palindromic repeat（CRISPR） interference（CRISPRi） specifically represses endogenous mi R-122 expression in Hep G2 cells.METHODS： Single-guide RNAs（sg RNAs; sgT1 and sgT2） targeting transcription start site（TSS） and TATA box in the region of miR- 122 promoter were designed. After co-transfecting Hep G2 cells with sgRNA and catalytically inactive dCas9- KRAB, the expression of mi R-122 was determined by quantitative Real-time PCR（qRT-PCR） to determine the more effective sgRNA. Different concentrations of sg RNA were tested in order to address whether CRISPRi was concentration dependent. The expression of miR-122 downstream target genes HOMX1 and Cyclin G1 was assessed by q RT-PCR and Western blot.RESULTS： Compared to the control group, CRISPRi mediated by sgT1 and sgT2 could repress endogenous mi R-122 expression by 5.5-fold（P〈0.01） and 3.7-fold（P〈0.01） in Hep G2 cells, respectively. The effect of CRISPRi was enhanced with increased concentration of sgRNA, and the miR- 1 2 2 expression was inhibited by 10.0-fold（P〈0.01） when the amount of lenti Guide-Puro-sgT1 was 500 ng. After endogenous mi R-122 expression was inhibited by CRISPRi, the expression of miR-122 downstream target genes HMOX1 and CyclinG 1 was upregulated.CONCLUSION： CRISPRi can specifically repress endogenous mi R-122 expression, which provides a novel therapeutic strategy against hepatitis C virus infection.