四川大学学报（医学版）2009，Vol.40Issue(6)：973-977,5.
靶向miR-221 siRNA表达载体的构建及有效靶序列的筛选和表达
Construction and Screening an Effective Anti-miR-221 RNAi Vector in vitro
摘要
Abstract
Objective To construct and screen an effective anti-mi.R-221 vector of siRNA. Methods Four hairpin structure of siRNA transcript templates targeting miR-221 and a negative control were synthesized, then ligated with pGCSIL-GFP vector and a pEGFP-miR-221 which express pre-mi.R-221 was also constructed. All the recombinants were sequenced. The confirmed pGCSIL-GFP recombinants by combining with pEGFP-miR-221 were transfected into 293T cells seperately. The expressed Flag protein was detected by Western blot to evaluate the inhibition effect of targeting sequences. Then a recombinant with the highest anti-miR-221 effect was screened and transfected into U87 glioma cell, and its anti-tumor effect was evaluated by MTT and FCM. Results The resulting recombinants were confirmed by sequencing which demonstrated that the recombinant plasmids contained the correct sequences of designed transcript templates. The results of Western blot indicated the expression of Flag of No 1 recombinant plasmid group was inhibited heavily with a 34. 3% expression level by compared with control group. The proliferation inhibition and induced apoptosis by this recombinant with an apoptosis ratio of 21. 89% in U87 cell were also observed. Conclusion The anti-miR-221 expression siRNA espression recombinants were constructed successfully, and one sequence with the highest inhibition efficiency was screened out, which could inhibit U87 cell proliferation and induce cell apoptosis, and could be used to suppress target gene for further study in tumor biology.关键词
miR-221/RNAi/DNA表达载体Key words
miR-221/RNAi/DNA expression vector分类
医药卫生引用本文复制引用
赵鹏,傅震,尤永平,陆小明,鲁艾林,刘宁..靶向miR-221 siRNA表达载体的构建及有效靶序列的筛选和表达[J].四川大学学报（医学版）,2009,40(6):973-977,5.基金项目
国家自然科学基金(批准号30672165)和江苏省医学重点人才项目(RC2007061)资助 （批准号30672165）
