miR-9对神经纤毛蛋白-1的靶向调控及其在辐射效应中的作用

目的：构建has-miR-9表达质粒和NRP1-3’UTR荧光素酶报告质粒，探讨miR-9对 NRP1的靶向调控作用及其在A549 细胞中的辐射效应。方法：利用生物信息学方法预测has-miR-9与NRP1-3’UTR的结合位点；将miR-9序列插入载体pcDNA-DEST47中构建真核表达质粒，同时构建NRP1-3’UTR的野生型和突变型荧光素酶报告质粒，并与pcDNA-DEST-miR-9质粒共转染至A549细胞，分析miR-9对其调控作用；miR-29b作为阴性对照，观察miR-9对NRP1的靶向作用。采用Western blot方法，验证miR-9对NRP1蛋白表达的抑制作用及照射后A549细胞NRP1蛋白表达变化；采用实时定量PCR方法检测10 Gy电离辐射照射后A549细胞miR-9表达量。结果：荧光素酶活性实验结果显示，miR-9可以显著下调野生型NRP1-3’UTR质粒的荧光素酶活性（t=3.906，P<0.05），而不影响突变型质粒的荧光素酶活性，同时证实miR-9以外的miRNA（miR-29b）不能抑制野生型NRP1-3’UTR质粒的荧光素酶活性。转染miR-9 mimic后，在A549细胞中，靶基因NRP1蛋白的表达受抑制。10 Gy照射后，A549细胞中miR-9表达量下调（t=37.319，P<0.05），而NRP1蛋白表达升高。结论：在A549辐射效应中miR-9通过靶向结合NRP1基因3’UTR，特异性调控NRP1蛋白表达。

Target regulation of miR-9 to the expression of NRP1 and its role in radiation effects

Objective: To explore the effect of miR-9 on the expression of NRP1 and its radiation effects in A549 cells. Methods: Bioinformatics was used to analyze the potential binding sites of has-miR-9 and NRP1-3'UTR. The miR-9 sequence was inserted into pcDNA-DEST-47 plasmid to construct the eukaryotic expression vector (pcDNA-DEST-miR-9) and to construct the NRP1 gene 3'UTR luciferase reporter plasmid (pEZX-MT05) at the same time. They were simultaneously transferred into A549 cells for analysis of the regulatory effect of miR-9 on the expression of NRP1. Meanwhile miR-29b was used as a negative control to observe whether or not NRP1 gene was a target of miR-9. After 10 Gy irradiation, the expression of NRP1, and the inhibitory effect of miR-9 on it was confirmed by Western blot assay. The expression of miR-9 was detected by real-time PCR. Results: It was found that miR-9 reduced the luciferase activity of NRP1-3'UTR wild plasmid (t=3.906, P<0.05) but not NRP1-3'UTR mutant plasmid. This luciferase activity was not inhibited by other types of miRNA (miR-29b). The expression of NRP1 protein in A549 cells was decreased after the cells were transfected with miR-9 mimic. After irradiation with dose of 10 Gy, the expression of miR-9 were decreased (t=37.319, P<0.05) and the expression of NRP1 protein were increased. Conclusions: miR-9 regulates the expression of NRP1 by targeting 3'UTR site of NRP1 gene in A549 cells.