miR-20a-5p调控成骨细胞分化相关靶基因的筛选及靶向关系验证

Screening of the target gene and verification of its targeting relationship with miR-20a-5p to regulate osteoblast differentiation

目的:筛选miR-20a-5p调控成骨分化的靶基因,深入研究miR-20a-5p调控成骨分化的分子机制。方法:通过靶基因预测软件预测miR-20a-5p的靶基因,将靶基因的3′UTR及点突变序列插入荧光素酶或绿色荧光蛋白(GFP)报告载体中,并与miR-20a-5p的模拟物或阴性对照共转染HEK-293T细胞,通过双荧光素酶实验和GFP抑制实验检测荧光素酶活性及GFP阳性细胞比例,确定miR-20a-5p与靶基因的靶向关系。结果:通过生物信息学预测到Smad7是miR-20a-5p的靶基因,miR-20a-5p在不同物种的Smad7基因3′UTR区都有保守的结合种子序列;荧光素酶、GFP报告基因及点突变载体经PCR及测序鉴定均正确;双荧光素酶活性检测结果显示:miR-20a-5p显著降低Smad73′UTR报告基因载体的荧光素酶活性;GFP抑制实验及流式细胞仪检测结果显示:miR-20a-5p可以显著降低GFP阳性细胞的比例。结论:miR-20a-5p可以直接靶作用于Smad73′UTR的种子序列,提示其可能通过直接靶向Smad7基因来发挥促进成骨分化的调控作用。

Objective:To screen the target gene of miR-20a-5p to regulate osteogenic differentiation,and study the molecular mechanism of miR-20a-5p regulating osteoblast differentiation.Methods:The target gene of miR-20a-5p was predicted by online software.The 3′UTR sequence and its mutant sequence of the target gene was cloned into the pMIR-REPORT and pMIR-GFP-REPORT.Then,HEK-293T cells were transfected with luciferase(or GFP)reporter vector and either miR-20a-5p mimics or miR-NC.Finally,determine the targeting relationship between miR-20a-5p and the target genes by luciferase activity and the GFP expression through dual luciferase reporter system and GFP repression assay.Results:As predicted by bioinformatics,Smad7 was the target gene of miR-20a-5p,and it contains a conserved seed sequence in 3′UTR element complementary to miR-20a-5p in different species.Results from PCR and DNA sequencing showed that the sequence of inserted Smad73′UTR in luciferase reporter constructions,GFP reporter constructions and all mutant constructions were correct.Dual luciferase reporter system showed that miR-20a-5p significantly repressed the reporter activity of Smad73′UTR reporter vector.GFP repression assay and FACS indicated that miR-20a-5p significantly reduced the proportion of GFP positive cells.Conclusion:miR-20a-5p can directly target the seed sequence of Smad73′UTR,suggesting that it may play a role in promoting osteogenic differentiation by targeting the Smad7 gene directly.