中文摘要：

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.

英文摘要：

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.