CRISPRi在体抑制肝脏miR-122的表达  

In vivo inhibition of liver mi R-122 expression by CRISPRi

作　　者：赵洪礼[1] 杨景玉[1] 李桂玲[1] 毛德华[1] 李洪运[2] 

机构地区：[1]山东省消化系统疾病防治中心,山东省医学科学院第三附属医院消化内科,山东省济宁市272033 [2]济宁市第一人民医院消化内科,山东省济宁市272033

出　　处：《世界华人消化杂志》2015年第29期4687-4693,共7页World Chinese Journal of Digestology

摘　　要：目的:探索CRISPR干扰(CRISPR interference,C R I S P R i)能否实现在体抑制肝脏m i R-122表达.方法:针对m i R-1 2 2启动子区设计sg RNA(sg T1和sg T2),并分别将其与无DNA切割活性仅保留识别活性的d Cas9-KRAB载体通过尾静脉流体力学法注射到8-10 wk龄小鼠,注射1、2、4 wk后通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)方法检测肝脏mmu-mi R-122的表达;设计不同的sg RNA浓度梯度,探索CRISPRi在体抑制肝脏mi R-122表达是否存在剂量依赖性;通过q RT-PCR及Western blot方法检测肝脏mi R-122靶分子HOMX1和Cyclin G1的表达变化.结果:在注射1 wk和2 wk后,sg T1介导的C R I S P R i在体抑制肝脏m i R-122的表达水平分别为23%(P<0.05)和16%(P<0.05);随sg RNA的剂量升高,肝脏mi R-122表达降低,当lenti Guide-Puro-sg T1质粒为120?g时,可将mi R-122的表达抑制约30%;CRIPSRi在体抑制肝脏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实现了在体抑制肝脏mi R-122的表达,为抗丙型肝炎病毒(hepatitis C virus)的在体治疗提供了新的策略.AIM：To investigate whether CRISPRi can repress miR-122 expression in the mouse liver in vivo.METHODS：sgRNAs（sgT1 and sgT2） were designed,targeting the region of mmumiR-122 promoter,sgRNAs（sgT1 or sgT2）and catalytically inactive dCas9-KRAB were delivered into 8-10-week-old mice by hydrodynamic tail-vein injection.The expression of mmu-miR-122 was detected at 1,2and 4 weeks after injection by quantitative realtime PCR（qRT-PCR） to determine the more effective sgRNA.Different concentrations of sgRNA were tested in order to address whether CRISPRi was concentration dependent in vivo.The expression of miR-122 downstream target genes HOMX1 and CyclinG1 was assessed by qRT-PCR and Western blot.RESULTS：Compared to the control group,CRISPRi mediated by sgT1 could repress miR-122 expression by 23%（P〈0.05） and 16%（P〈0.05） in the mouse liver at 1 and 2 weeks after injection,respectively.The effect of CRISPRi was enhanced with increased concentrations of sgRNA.After mmu-miR-122 expression in the mouse liver was inhibited by CRISPRi in vivo,the expression of miR-122 downstream target genes HMOX1 and CyclinG1 was up regulated.CONCLUSION：CRISPRi can specially repress miR-122 expression in the liver in vivo,which provides a novel therapeutic strategy against hepatitis C virus（HCV） infections.

关 键 词：CRISPRi 在体基因修饰 MI R-122 肝脏 

分 类 号：R512.63[医药卫生—内科学]