miR-29a靶基因VEGF-A的生物信息学预测及鉴定

The prediction and identification of novel mi R-29a target gene VEGF-A

目的预测并鉴定miR-29a的靶基因血管内皮生长因子VEGF-A。方法采用生物信息学技术,预测miR-29a与VEGF-A mRNA 3'UTR结合位点;分别采用RT-q PCR、Western blot技术检测过表达miR-29a后,BGC-823细胞内VEGF-A表达水平的变化;采用双荧光素酶实验,验证miR-29a与VEGF-A mRNA 3'UTR的结合位点。结果生物信息学技术预测显示miR-29a可稳定结合于VEGF-A mRNA 3'UTR;miR-29a过表达后可明显抑制BGC-823细胞VEGF-A mRNA 及蛋白表达水平;双荧光素酶实验证实,与阴性对照相比,miR-29a拟似物(mimics)可通过稳定结合于VEGF-A mRNA 3'UTR抑制荧火虫荧光素酶活性(0.48±0.08 vs 1.00±0.22,P<0.05)。结论 miR-29a通过直接结合于靶基因VEGF-A mRNA 3'UTR抑制VEGF-A蛋白表达。

[Objective] To predict and identify the target gene VEGF-A of miR-29a. [Methods] The bioinformatics analysis was used to predict the binding sites between miR-29a and VEGF-A mRNA 3＇UTR. RT- qPCR and western-blot were used to examine the expression level of VEGF-A after over-expression of miR-29a in BGC-823 cells. The dual lueiferase reporter assay identified the binding site between miR-29a and VEGF-A mRNA 3＇UTR. [Results] The bioinformatics analysis found the stable binding sites between miR-29a and VEGF-A mRNA 3＇UTR. Over-expression of miR-29a in BGC-823 cells could significantly suppress the expression of VEGF- A at both mRNA and protein level. The dual luciferase reporter assay verified that the miR-29a mimics could suppress the activity of firefly luciferase significantly by stably binding to the VEGF-A mRNA 31UTR （0.48±0.08 vs. 1.00±0.22, P 〈0.05）. [Conclusion] miR-29a could stably bind to VEGF-A mRNA 3＇UTR and suppress the expression of VEGF-A, which makes miR-29a a promising target in tumor gene therapy.