针对人STAT1基因的RNA干扰有效靶点的设计与筛选

Design and selection of effective RNA interference target for STAT1 gene

目的:设计并筛选针对人STAT1基因有效的RNA干扰靶点。方法:根据人STAT1基因序列,设计3个干扰序列,将oligo退火成双链并连接穿梭载体pLKO-1-EGFP-puro-shRNA,构建shRNA慢病毒载体质粒,并转化至感受态细胞DH5a,进行测序验证。在脂质体介导下转染293T细胞,包装生产慢病毒。慢病毒载体转染人肝癌细胞MHCC97L的STAT1过表达瘤株(MHCC97L-stat1),用Real-timePCR检测干扰效果。结果:测序证实3个STAT1基因RNAi病毒载体质粒构建成功;慢病毒载体经293T细胞包装成功;3个慢病毒载体转染人肝癌细胞MHCC97L-stat1瘤株48h后,STAT1基因在mRNA水平表达均受到抑制,其中MHCC9-L-stat1-shRNA-1序列效果最佳,对STAT1基因表达的干扰效率可达81.4%。结论:成功构建并筛选了人STAT1基因RNAi慢病毒载体及有效靶点,为进一步深入研究STAT1基因与抗肿瘤药物药效关系奠定基础。

Objective： To design and select effective RNA interference（RNAi） target for STAT1 gene in human. Methods： Based on the sequence of STAT1 gene in human, we designed three interference sequences to make oligo anneal into double chains connected with shuttle vector pLKO-1-EGFP-puro-shRNA. Then we constructed lentiviral vector plasmid of shRNA and transformed them into competent cells DH5 which we verified by sequencing. Under the liposome mediation 293T cells were transfected to achieve the lentivirus packaging production. STAT1 of MHCC97L in human hepatoma cells which were transfected by lentiviral vectors showed overexpression of tumor cell strains（MHCC97L-stat1）. We detected the validity of RNA interference by Real-time PCR, Results： It is confirmed by sequencing that three tentiviral vectors plasmid of STAT1 RNAi were constructed successfully.The packaging production of lentiviral vectors transfected by 293T cells was successful.48h after these three lentivirat vectors transfected MHCC97L of human hepatoma cells, the expression of STAT1 were all suppressed in the mRNA level, in which MHCC9-L-statl-shRNA-1 line had the best effection and the efficiency of RNA interference was up to 81 4% Conclusion： The lentiviral vectors of STAT1 RNAi in human were constructed suc- cessfully, and we also selected out the effective target It laid the foundation of further study on the relationship between STAT1 gene and antineoptastic drug.