【摘要】 目的通过构建成纤维细胞生长因子-21基因(fibroblast growth factor 21,FGF-21)荧光素酶报告基因载体,观察has-miR-577和has-miR-583对FGF-21基因的靶向调控。方法利用生物学信息网站对has-miR-577和has-miR-583靶向结合FGF-21 3′UTR区的位点进行分析,设计合成包含与has-miR-577和has-miR-583结合序列及其突变序列的FGF-21基因片段,构建野生型(psiCHECK2-FGF-21)和突变型(psiCHECK2-FGF-21-mut)FGF-21双荧光素酶报告基因载体。进行双酶切电泳和测序鉴定后,FGF-21野生型与突变型双荧光素酶报告基因载体分别+〔hsa-miR-577模拟物、hsa-miR-583模拟物和miR无义序列阴性对照(miR negative control,miR-NC)〕转染293T细胞,检测荧光素酶活性,观察has-miR-577和has-miR-583对FGF-21表达的影响。结果双酶切电泳和测序结果显示,野生型(psiCHECK2-FGF-21)和突变型(psiCHECK2-FGF-21-mut)基因载体片段大小、序列结果与实验预期一致,载体构建成功。has-miR-577和has-miR-583可抑制野生型FGF-21载体的荧光表达(P<0.05),而对突变型FGF-21载体的荧光表达无抑制作用。结论 has-miR-577和has-miR-583可以靶向调控FGF-21的表达。更多还原

【Abstract】 Objective To determine the targeted regulating role of has-miR-577 and has-miR-583 on the expression of fibroblast growth factor 21(FGF-21)based on a constructed luciferase reporter FGF-21 gene vector.Methods The site of has-miR-577 and has-miR-583 target genes FGF-21 were predicted by the bioinformatics analyzing tools online.FGF-21 gene fragments,combined with has-miR-577 or has-miR-583 sequences and mutant sequences,were designed and synthesized.The wild type(psiCHECK2-FGF-21)and mutant(psiCHECK2-FGF-21-mut)luciferase reporter gene carriers were constructed.The relevant plasmids 〔hsa-miR-577 mimics,hsa-miR-583 mimics or miR negative control(miR-NC)〕and luciferase reporter gene carrier(wild type or mutant)were cotransfected into 293 Tcells.The luciferase reporter system was used to detect the luciferase activity.The effects of has-miR-577 and has-miR-583 on the expression of FGF-21 were observed.Results The double enzyme electrophoresis and sequencing results showed that the gene fragment size and sequences of the wild type(psiCHECK2-FGF-21)and mutant(psiCHECK2-FGF-21-mut)carriers met expectations of the experiment.The luciferase assays revealed that has-miR-577 and has-miR-583 significantly diminished luciferase activity from the reporter vector containing 3′UTR of FGF-21(P<0.05),whereas no suppression of luciferase activity was found in the mutant(psiCHECK2-FGF-21-mut).Conclusion FGF-21 gene can be targeted by has-miR-577 and has-miR-583.更多还原