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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。
Objective To investigate the regulation of FGF-21 gene expression by has-miR-577 and has-miR-583 by constructing luciferase reporter gene vector of fibroblast growth factor 21 (FGF-21). Methods The site of 3’UTR region of has-miR-577 and has-miR-583 that binds to FGF-21 was analyzed by the bioinformatics website. (PsiCHECK2-FGF-21) and mutant (psiCHECK2-FGF-21-mut) FGF-21 dual luciferase reporter gene vectors were constructed based on the FGF-21 gene fragment of the FGF-21 gene sequence and its mutated sequence. After double enzyme digestion and sequencing, FGF-21 wild-type and mutant dual luciferase reporter vectors + [hsa-miR-577 mimics, hsa-miR-583 mimics and miR nonsense sequence negative controls miR-577 and miR-583 on the expression of FGF-21 in 293T cells were detected by flow cytometry. Results The results of double enzyme digestion electrophoresis and sequencing showed that the size and sequence of wild-type (psiCHECK2-FGF-21) and mutant (psiCHECK2-FGF-21-mut) gene fragments were consistent with the experimental results and the vector was successfully constructed. has-miR-577 and has-miR-583 can inhibit the expression of wild-type FGF-21 vector (P <0.05), but did not inhibit the expression of mutant FGF-21 vector. Conclusion HAS-miR-577 and has-miR-583 can target FGF-21 expression.