miR-9 对NRP1 的靶向调控及其在辐射效应中的作用研究
张海芹，董娟聪，金霖霖，高辉，邵立虹，刘丽波，金顺子
基金项目：国家自然科学基金（30870584,81371890）；教育部高等学校博士学科点专项科研基金（20120061110063）
作者简介：张海芹（1987-），女，硕士研究生，主要从事辐射肿瘤学研究
通信联系人：金顺子（1965-），女，教授，主要从事辐射免疫学与辐射肿瘤学的研究. 

 （吉林大学公共卫生学院 卫生部放射生物学重点实验室，长春 130021）
摘要：目的：构建hsa-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hsa-miR-9 与 NRP1-3 ′ -UTR 的结合位点； 将PCR 扩增的miR-9 前体序列和
pcDNA-DEST47 载体连接，构建pcDNA-DEST47-miR-9 表达质粒；将PCR 扩增的 NRP1-310 ′ UTR 序列 和pEZX-MT05 载体经Acc65 I 和Xho I 双酶切后连接， 构建pEZX-MT05-NRP1-3′UTR 表达质粒；以此质粒为模板，根据突变体引物序列扩增 NRP1-3
′UTR 突变序列，构建pEZX-MT05- NRP1-3′UTRMUT 表达质粒。采用双荧光素酶检测法验证miR-9 与NRP1 的靶向关系；采用实时定量 PCR 及 Western blot 分别检测10 Gy照射后miR-9 的表达水平及对 NRP1 蛋白表达的靶向抑制作用。结果：成功构建 pcDNA-DEST47-miR-9、pEZX-MT05-NRP1- 3′UTR 及pEZX-MT05-NRP1-3′UTR MUT表达质粒，并通过酶切及基因测序得到鉴定；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。实时定量 PCR 结果显示，miR-9 的表达水平在10 Gy 照射后明显降低（t=37.319，p<0.01），加入miR-9 mimic 虽然可以抑制辐射引起的miR-9 的表达下调，但还是低于单纯mimic 组；Western blot 结果显示，miR-9 对NRP1 的蛋白表达具有抑制作用，但加入miR-9 mimic 可以阻止辐射诱导的NRP1 表达上调，而加入miR-9 inhibitor 时却其表达上调。结论：证实miR-9 通过靶向结合 NRP1 基因3'UTR，特异性调控 NRP1 蛋白表达；10 Gy X 射线使A549 细胞中hsa-miR-9 表达下调，从而NRP1 蛋白表达上调，提示在 A549 辐射效应中miR-9 对NRP1 具有靶向调控作用。
关键词：miR-9；NRP1；A549；靶向调控；电离辐射
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Target regulation of miR-9 on the expression of NRP1 and its role in the study of irradiation effects
ZHANG Haiqin, DONG Juancong, JIN Linlin, GAO Hui, SHAO Lihong, LIU Libo,
JIN shunzi
(College of Public Medicine, Key Laboratory of Radiobiology, Ministry of Health,jilin University,ChangChun 130021)
 Abstract: Objective has-miR-9 recombinant plasmid and NRP1-3'UTR luciferase reporter plasmid was constructed and to explore the effect of miR-9 on the expression of NRP1 and its irradiation effects in A549 cells. Methods Bioinformatics was used to analyze the potential binding sites of sa-miR-9 and NRP1-3′UTR. The precursor of miR-9 was amplified by PCR and the PCR products were cloned into pcDNA-DEST47 vector to construct pcDNA-DEST47-miR-9 expression plasmid. NRP1-3′UTR was amplified by PCR and the PCR products were cloned into Acc65 I and Xho I digestion pEZX-MT05 vector to construct pEZX-MT05-NRP1-3′-UTR expression plasmid ; pEZX-MT05-NRP1-3'UTRMUT expression plasmid was amplifie according to the mutant sequence of pEZX-MT05-NRP1-3'-UTR ; The effect of miR-9 interaction with the 3’-UTR of NRP1 on luciferase activity was detected with a dual luciferase assay system ；the expression level of NRP1 45 protein affected by miR-9 was detected by Western blotting and the level of miR-9 was detected by Real-time PCR after 10 Gy X rays. Results pcDNA-DEST47-miR-9、pEZX-MT05-NRP1-3′UTR and pEZX-MT05-NRP1-3′UTRMUT plasmids were successfully constructed ; and has been identified by restriction analysis and DNA sequencing ; Luciferase reporter experiments suggested that miR-9 could reduce the luciferase activity of wild plasmid (t = 3.906, p<0.05), without affecting the luciferase activity of the mutant plasmid and the luciferase activity was not inhibited in other types of miRNA( miR-29b )；The real-time PCR suggested that the level of miR-9 was significantly reduced after 10 Gy X rays （t=37.319，p<0.01）; While adding miR-9 mimic can inhibit the expression of radiation-induced down-regulation of miR-9, but still lower than that mimic group ；Western blot suggested that the level of NRP1 was inhibited by miR-9 , while the upregulation of NRP1 induced by irradiation could be suppressed by miR-9 mimic. Conclusion Theses results suggested that miR-9 regulates the expression of NRP1 by targeting the complementary sites and specific regulation of NRP1 protein expression , the level of has-miR-9 was downregulated while the protein level of
NRP1was upregulate after 10 Gy Xrays , miR-9 plays a role in the process of target regulation of  irradiation on expression of NRP1 protein in A549.I
Key words: miR-9; NRP1; A549; Targeted regulation; Ionizing radiation